Methods for synthesizing anticoagulant polysaccharides

ABSTRACT

The present invention includes methods for preparing anticoagulant polysaccharides using several non-naturally occurring, engineered sulfotransferase enzymes that are designed to react with aryl sulfate compounds instead of the natural substrate, PAPS, to facilitate sulfo group transfer to polysaccharide sulfo group acceptors. Suitable aryl sulfate compounds include, but are not limited to, p-nitrophenyl sulfate or 4-nitrocatechol sulfate. Anticoagulant polysaccharides produced by methods of the present invention comprise N-, 3-O-, 6-O-sulfated glucosamine residues and 2-O sulfated hexuronic acid residues, have comparable anticoagulant activity compared to commercially-available anticoagulant polysaccharides, and can be utilized to form truncated anticoagulant polysaccharides having a reduced molecular weight.

CROSS-REFERENCE TO RELATED APPLICATIONS

The instant application is a continuation of U.S. patent applicationSer. No. 17/376,354, filed on Jul. 15, 2021, which is acontinuation-in-part of International Application No. PCT/US2021/007429,filed Jul. 9, 2020, which claims the benefit of U.S. ProvisionalApplication No. 62/871,980, filed on Jul. 9, 2019, and 63/033,687, filedon Jun. 2, 2020, the disclosures of which are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to methods for synthesizing anticoagulantpolysaccharides using engineered, non-natural sulfotransferase enzymesthat are designed to react with aryl sulfate compounds as sulfo groupdonors.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a sequence listing inelectronic format. The sequence listing is provided as a file entitled“OPT-002XRT-CT_sequence_disclosure.xml” created on Aug. 23, 2022, andwhich is 129,141 bytes in size. The information in electronic format ofthe sequence listing is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Heparins and other anticoagulant polysaccharides are a class ofcompounds that are commonly prescribed as drugs in clinical settings toprevent blood clotting. Typically, these compounds are isolated andpurified from the internal organs of animals, such as pigs and cows.However, because of recent disruptions in the worldwide supply due topotential contamination of heparin (over 200 people died as a result ofcontaminated compounds in 2007 in the United States alone) andgeopolitical tensions with global suppliers, there has been a recentpush to synthesize anticoagulant polysaccharides in vitro.

Within the animal, sulfated polysaccharides, including heparin, aresynthesized by the catalytic transfer of sulfate functional groups, alsocalled “sulfo groups”, from a sulfo group donor to a polysaccharide,which acts as a sulfo group acceptor. Each sulfo group transfer iscatalyzed by a sulfotransferase enzyme, and there are often multiplesulfotransfer reactions catalyzed by multiple sulfotransferase enzymesto ultimately arrive at each sulfated polysaccharide product.Sulfotransferases are nearly ubiquitous in nature, and they exist innearly all types of organisms, including bacteria, yeast, and animals,including humans. Similarly, sulfotransferase enzymes play an integralrole in the sulfation of a wide array of sulfo group acceptors,including many types of steroids, polysaccharides, proteins,xenobiotics, and other molecules.

There are several polysaccharides that can be utilized as sulfo groupacceptors, including, for example, dermatan, keratan, heparosan, andchondroitin. In particular, heparosan comprises repeating disaccharideunits of 1→4 glycosidically-linked, glucuronic acid and N-acetylatedglucosamine ([β(1,4)GlcA-α(1,4)GlcNAc]_(n)) residues, any of which canbe further modified by one or more enzyme-catalyzed deacetylation,sulfation, or epimerization reactions. Sulfation of heparosan-basedpolysaccharides can be catalyzed by up to four sulfotransferase enzymesto form heparan sulfate (HS), and when performed in a particular orderalong with deacetylation of one or more glucosamine residues andepimerization of one or more glucuronic acid residues, can be utilizedto form heparin.

there are only a couple of molecules that can be utilized bysulfotransferase enzymes as sulfo group donors. The nearly ubiquitoussulfo group donor, including for each of the four HS sulfotransferases,is 3′-phosphoadenosine 5′-phosphosulfate (PAPS). These in vivo systemshave evolved to exclusively utilize PAPS because it has a shorthalf-life and can readily be synthesized and metabolized, as needed, bythe organism. However, that same short half-life renders PAPS to beunsuitable for most in vitro syntheses, particularly in large scalesyntheses, that utilize sulfotransferases because it can readilydecompose into adenosine 3′,5′-diphosphate, which actively inhibits thesulfotransferases' biological activity.

Aryl sulfate compounds, such as p-nitrophenyl sulfate (PNS) and4-methylumbelliferyl sulfate (MUS) have been identified as cheap,widely-available compounds that can be useful as sulfo donors with avery limited number of sulfotransferases to synthesize certain smallmolecule products (see Malojcic, G., et al. (2008) Proc. Nat. Acad. Sci.105 (49):19217-19222 and Kaysser, L., et al., (2010) J. Biol. Chem. 285(17):12684-12694, the disclosures of which are incorporated by referencein their entireties). Yet, only a small number of bacterialsulfotransferases have been shown to react with aryl sulfate compoundsas sulfo group donors, and none of these react with polysaccharides, letalone heparosan-based polysaccharides, as sulfo group acceptors. As aresult, when sulfotransferases are used in the in vitro synthesis ofsulfated polysaccharides, PAPS must be included in the reaction mixtureto effectively catalyze sulfo group transfer, and aryl sulfate compoundscan only be used indirectly, to repopulate the system with PAPS (seeU.S. Pat. No. 6,255,088, the disclosure of which is incorporated byreference in its entirety).

Consequently, there is a need to develop facile methods of synthesizingsulfated polysaccharides in vitro, particularly heparin, withoututilizing PAPS as the sulfo group donor. In particular, the developmentof sulfotransferase enzymes that are capable of both reacting with arylsulfate compounds as sulfo group donors and with heparosan-basedpolysaccharides as sulfo group acceptors would present a large stepforward toward the development of large-scale syntheses of heparin invitro.

SUMMARY OF THE INVENTION

The present invention provides methods for producing sulfatedpolysaccharides, particularly heparin and heparin derivatives, in vitrousing non-naturally occurring sulfotransferase enzymes that have beenengineered to catalyze the transfer of sulfo groups from aryl sulfatecompounds as sulfo group donors to react with polysaccharides as sulfogroup acceptors. According to the present invention, the polysaccharidescan be heparosan-based polysaccharides that can be utilized to formheparin and other sulfated polysaccharides that possess anticoagulantactivity. According to the present invention, heparin synthesized by themethods described herein can be prescribed and administered in aclinical setting to prevent blood clotting.

In an aspect of the present invention, a sulfated polysaccharide productcan be synthesized enzymatically by a method comprising the steps of:(a) providing a polysaccharide; (b) providing an aryl sulfate compound;(c) providing an engineered sulfotransferase enzyme configured torecognize, bind, and react with the aryl sulfate compound as a sulfogroup donor, and with the polysaccharide as a sulfo group acceptor; (d)forming a reaction mixture by combining the engineered sulfotransferaseenzyme with the polysaccharide and the aryl sulfate compound; and (e)catalyzing the enzymatic transfer of a sulfo group from the aryl sulfatecompound to the polysaccharide, using the engineered sulfotransferaseenzyme, to form the sulfated polysaccharide product. According to thepresent invention, the polysaccharide can be a heparosan-basedpolysaccharide derived from heparosan, [β(1,4)GlcA-α(1,4)GlcNAc]_(n), inwhich GlcA is glucuronic acid and GlcNAc is N-acetyl glucosamine.Heparosan-based polysaccharides comprise repeating dimers of 1→4glycosidically-linked hexuronic acid and glucosamine residues, whereineach hexuronic acid is either glucuronic acid (GlcA, above) or iduronicacid (IdoA), and each glucosamine residue can either be N-acetylated,N-sulfated, or N-unsubstituted. Heparosan-based polysaccharides in whichat least one of the glucosamine residues is N-unsubstituted can also becalled N-deacetylated heparosan. Further, in various embodiments, any ofthe GlcA or IdoA residues can be sulfated at the 2-O position, and/orany of the glucosamine residues can be sulfated at the N-, 6-O, or 3-Oposition, prior to reacting with an engineered sulfotransferase enzyme.Heparosan-based polysaccharides that contain at least one sulfate groupin any of the above positions within a hexuronic acid or glucosamineresidue can also be called heparan sulfate (HS).

According to the present invention, and useful in combination with anyone or more of the above aspects and embodiments, a sulfatedpolysaccharide product formed in a first sulfotransfer reaction can beutilized as a sulfo group acceptor in a subsequent reaction with anothersulfotransferase enzyme, which can either be performed in the samereaction mixture as the first sulfotransfer reaction, or in a separatereaction mixture after isolating the sulfated polysaccharide product andcombining it with a sulfo group donor and a sulfotransferase enzyme. Invarious embodiments, a plurality of sulfotransfer reactions can becarried out, either sequentially or simultaneously, on a singleheparosan-based polysaccharide, including at least two, at least three,or at least four sulfotransfer reactions. Each of the plurality ofsulfotransfer reactions on a heparosan-based polysaccharide can becatalyzed by at least two, at least three, or up to foursulfotransferase enzymes. In various embodiments, at least one, andpreferably all, of the sulfotransfer reactions are catalyzed by anengineered sulfotransferase enzyme which recognizes, binds, and reactswith the aryl sulfate compound as a sulfo group donor. In furtherembodiments, at least one, and preferably all, of the sulfotransferreactions are carried out in reaction mixtures that contain only an arylsulfate compound as a sulfate donor, and do not contain PAPS.

In another aspect of the invention, each engineered sulfotransferaseenzyme comprises several amino acid mutations made within the activesite of a corresponding natural sulfotransferase enzyme, in order toconvert the enzyme's biological activity from reacting with PAPS as thesulfo group donor to reacting with an aryl sulfate compound as a sulfogroup donor. However, in various embodiments, each engineeredsulfotransferase enzyme retains the natural enzyme's biological activitywith its particular sulfo acceptor polysaccharide. As a non-limitingexample, a natural HS hexuronyl 2-O sulfotransferase (2OST), which has abiological activity in which the enzyme reacts with PAPS as a sulfogroup donor and N-sulfated heparosan as a sulfo group acceptor, can bemutated in multiple amino acid positions to generate an engineeredsulfotransferase enzyme that recognizes, binds, and reacts with an arylsulfate compound as a sulfo group donor, but that still reacts withN-sulfated heparosan as a sulfo group acceptor. Such engineered arylsulfate-dependent 2OST enzymes, and others, are described in furtherdetail below.

In another aspect of the invention, an N-, 2-O-, 3-O-, 6-O-sulfatedheparan sulfate (N,2,3,6-HS) product can be synthesized, the methodcomprising the following steps: (a) providing a starting polysaccharidereaction mixture comprising N-deacetylated heparosan; (b) combining thestarting polysaccharide reaction mixture with a reaction mixturecomprising a sulfo group donor and a first sulfotransferase enzymeselected from the group consisting of a glucosaminyl N-sulfotransferaseenzyme (NST), a 2OST enzyme, and a glucosaminyl 6-O sulfotransferase(6OST) enzyme, to form a first sulfated polysaccharide; (c) combiningthe first sulfated polysaccharide with a reaction mixture comprising asulfo group donor and a second sulfotransferase enzyme, wherein thesecond sulfotransferase enzyme is one of the two enzymes that were notselected in step (b), to form a second sulfated polysaccharide; (d)combining the second sulfated polysaccharide with a reaction mixturecomprising a sulfo group donor and a third sulfotransferase enzyme,wherein the third sulfotransferase enzyme is the enzyme that was notselected in step (b) or step (c), to form a third sulfatedpolysaccharide; and (e) combining the third sulfated polysaccharide witha reaction mixture comprising a sulfo group donor and a glucosaminyl 3-Osulfotransferase (3OST) enzyme, to form the N,2,3,6-HS product; wherein(i) at least one of the sulfotransferase enzymes is an engineeredsulfotransferase enzyme that is dependent on reacting with an arylsulfate compound as a sulfo group donor to catalyze a sulfotransferreaction, and (ii) in a reaction mixture comprising an engineeredsulfotransferase enzyme, the reaction mixture comprises an aryl sulfatecompound as a sulfo group donor. In various embodiments, the firstsulfotransferase enzyme is an NST enzyme, the second sulfotransferaseenzyme is a 2OST enzyme, and the third sulfotransferase enzyme is a 6OSTenzyme.

In another aspect of the invention, methods to synthesize an N,2,3,6-HSproduct can comprise the following steps: (a) providing a startingpolysaccharide reaction mixture comprising N-sulfated heparosan; (b)combining the starting polysaccharide reaction mixture with a reactionmixture comprising a sulfo group donor and a first sulfotransferaseenzyme selected from the group consisting of a 2OST enzyme and a 6OSTenzyme, to form a first sulfated polysaccharide product; (c) combiningthe first sulfated polysaccharide product with a reaction mixturecomprising a sulfo group donor and a second sulfotransferase enzyme,wherein the second sulfotransferase enzyme is the enzyme that was notselected in step (b), to form a second sulfated polysaccharide product;and (d) combining the second sulfated polysaccharide product with areaction mixture comprising a sulfo group donor and a 3OST enzyme, toform the N,2,3,6-HS product; wherein (i) at least one of thesulfotransferase enzymes is an engineered sulfotransferase enzyme thatis dependent on reacting with an aryl sulfate compound as a sulfo groupdonor to catalyze a sulfotransfer reaction, and (ii) in a reactionmixture comprising an engineered sulfotransferase enzyme, the reactionmixture comprises an aryl sulfate compound as the sulfo group donor. Invarious embodiments, the first sulfotransferase enzyme is the 2OSTenzyme, and the second sulfotransferase enzyme is the 6OST enzyme.

In various embodiments, the starting polysaccharide reaction mixturecomprising N-sulfated heparosan can be provided by combiningN-deacetylated heparosan, a sulfo group donor, and an NST enzyme into areaction mixture. In various embodiments, the NST enzyme is anengineered sulfotransferase, which reacts with an aryl sulfate compoundas a sulfo group donor in the absence of PAPS. In various embodiments,the N-sulfated heparosan can be provided by combining a naturalglucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzyme, PAPS, andheparosan.

In various embodiments, the step of providing the startingpolysaccharide reaction mixture can comprise the chemical synthesis ofN-sulfated heparosan, comprising the following sub-steps: (i) providinga precursor polysaccharide composition comprising heparosan; (ii)combining the precursor polysaccharide composition with a reactionmixture comprising a base, preferably lithium hydroxide or sodiumhydroxide, for a time sufficient to N-deacetylate at least one of theN-acetylated glucosamine residues within the heparosan, forming anN-deacetylated heparosan composition; and (iii) combining theN-deacetylated heparosan composition with a reaction mixture comprisingan N-sulfation agent, thereby forming the N-sulfated heparosan.

In various embodiments, the step of providing the precursorpolysaccharide composition comprising heparosan can further comprise thesub-step of isolating heparosan from a bacterial or eukaryotic cellculture, preferably a bacterial cell culture, and more preferably abacterial cell culture comprising bacteria selected from the groupconsisting of the K5 strain of Escherichia coli (E. coli) and the BL21strain of E. coli. Heparosan can be isolated from E. coli as apolydisperse mixture of polysaccharides having a weight-averagemolecular weight of at least 10,000 Da, and up to at least 500,000 Da.

Treating heparosan with a base, such as lithium hydroxide or sodiumhydroxide, removes acetyl groups from N-acetyl glucosamine residues,forming N-unsubstituted glucosamine residues that can subsequently beN-sulfated. In various embodiments, precursor polysaccharides can betreated with a base for a time sufficient to reduce the relative numberof N-acetylated glucosamine residues to a desired level. The reactiontime can be dependent on factors such as the average molecular weight ofthe heparosan within the precursor polysaccharide composition, theN-acetyl glucosamine content of the heparosan prior to reacting with thebase, the desired N-acetyl content within the N-deacetylated heparosancomposition, and the concentration and identity of the base itself. Invarious embodiments, the time sufficient to N-deacetylate the heparosanwithin the precursor polysaccharide composition can be the timesufficient to form an N-deacetylated heparosan composition in which lessthan 60%, down to less than 5%, preferably in the range of 12% to 18%,and more preferably 15%, of the glucosamine residues remainN-acetylated.

Additionally, treating the precursor polysaccharide composition with abase to reduce the number of N-acetylated glucosamine residues can alsohave the effect of depolymerizing the heparosan, causing theN-deacetylated heparosan composition to have a lower average molecularweight relative to the precursor polysaccharide composition.Accordingly, in various embodiments, the precursor polysaccharidecomposition can be treated with a base for a time sufficient to form anN-deacetylated heparosan composition having a desired average molecularweight. As with above, the reaction time can depend on several factors,including the average molecular weight of the heparosan within theprecursor polysaccharide composition, and the desired average molecularweight of the polysaccharides within the N-deacetylated heparosancomposition itself. In various embodiments, the time sufficient toN-deacetylate the heparosan within the precursor polysaccharidecomposition can be the time sufficient to form an N-deacetylatedpolysaccharide composition having a weight-average molecular weight in arange from 1,500 Da to 100,000 Da, for example, from at least 1,000 Da,up to 20,000 Da, or from at least 9,000 Da, and up to 12,500 Da.

In various embodiments, once the N-deacetylated heparosan is formed, theresulting N-unsubstituted glucosaminyl residues can then receive a sulfogroup from an N-sulfation agent, such as, for example, an engineered ornatural NST enzyme. In various embodiments, one or more of theN-unsubstituted glucosamine residues within N-deacetylated heparosan canbe chemically N-sulfated. In various embodiments, chemical N-sulfationcan either supplement or replace enzymatic N-sulfation catalyzed by anNST enzyme. A non-limiting example of a chemical N-sulfation agent cancomprise a reaction mixture comprising a sulfur trioxide-containingcompound or adduct, particularly a sulfur trioxide-trimethylamineadduct.

In various embodiments, by either chemical and/or enzymatic N-sulfation,at least about 10%, and up to at least about 95%, of the glucosaminylresidues within N-deacetylated heparosan are N-sulfated by theN-sulfation agent, prior to subsequently being sulfated at any of the2-O, 3-O, or 6-O positions.

In various embodiments, during a synthesis of an N,2,3,6-HS productaccording to any of the methods described herein, the 3-O sulfation ofthe heparosan-based polysaccharide can be catalyzed after the 2-Osulfation step to form an N,2,3-HS product, followed by 6-O sulfation toform the N,2,3,6-HS product. Additionally, within any of thesulfotransfer reaction steps within methods described herein, reactionmixtures that do not comprise an engineered sulfotransferase enzyme cancomprise PAPS and a natural HS sulfotransferase enzyme that possessesbiological activity with PAPS as the sulfo group donor. In variousembodiments, even if one or more of the NST enzyme, 2OST enzyme, and6OST enzyme used to form an N,2,3,6-HS product is a naturalsulfotransferase enzyme, the synthesis is completed using an engineered3OST enzyme, using an aryl sulfate compound as a sulfo group donor inthe absence of PAPS. In various embodiments, the sulfotransferase enzymein all sulfotransfer steps in the synthesis of an N,2,3,6-HS product isan engineered sulfotransferase enzyme, in which the sulfo group donor ineach step consists of one or more aryl sulfate compounds and thesulfotransferase reaction takes place in the absence of PAPS. In variousembodiments, reaction mixtures for one or more of the sulfotransferreactions can be combined into a single reaction vessel, or “pot.” Inother embodiments, each of the sulfotransfer reactions can be conductedsequentially, in separate reaction vessels.

In various embodiments, aryl sulfate compounds that can be utilized assulfo donors are organosulfates that comprise a sulfo group covalentlybound to an aromatic moiety, bound together by a sulfate ester linkagecomprising a C—O bond. Non-limiting examples of aryl sulfate compoundsthat are suitable substrates with the engineered enzymes of the presentinvention include p-nitrophenyl sulfate (PNS), 4-methylumbelliferylsulfate (MUS), 7-hydroxycoumarin sulfate, phenyl sulfate, 4-acetylphenylsulfate, indoxyl sulfate, 1-naphthyl sulfate, 2-naphthyl sulfate(2NapS), and 4-nitrocatechol sulfate (NCS). In various embodiments,engineered enzymes utilized in accordance with any of the methods of thepresent invention can recognize, bind, and react with PNS. In someembodiments, PNS can be used as the aryl sulfate compound in everysulfotransfer reaction during the synthesis of the N,2,3,6-HS product.According to the present invention, engineered enzymes utilized inaccordance with any of the methods of the present invention canrecognize, bind, and react with NCS. In some embodiments, NCS can beused as the aryl sulfate compound in every sulfotransfer reaction duringthe synthesis of the N,2,3,6-HS product. According to the presentinvention, a single engineered enzyme utilized in accordance with any ofthe methods of the present invention can recognize, bind, and react withmultiple aryl sulfate compounds.

In various embodiments, each of the engineered sulfotransferase enzymesutilized in the synthesis of an N,2,3,6-HS product according to any ofthe methods described herein can be selected to react with the same arylsulfate compound as a sulfo group donor. In other embodiments, one ormore of the engineered sulfotransferase enzymes can have a biologicalactivity with different aryl sulfate compounds than other enzymesutilized in the same synthesis of an N,2,3,6-HS product. As anon-limiting example, an N,2,3,6-HS product can be synthesized usingsome engineered enzymes that react with NCS as a sulfo group donor,while other engineered enzymes within the same synthesis react with PNSas a sulfo group donor, and in syntheses in which multiple sulfotransferreactions are carried out in a single reaction mixture, both PNS and NCScan be included within the reaction mixture.

In various embodiments, within any reaction mixture or compositioncomprising heparosan-based polysaccharides used as starting materials orformed as products while practicing any of the methods of the presentinvention, including but not limited to precursor polysaccharides,starting polysaccharides, sulfated polysaccharide products, and/orheparin, the polysaccharides can be present as a polydisperse mixture ofvaried chain lengths, molecular weights, N-acetylation, and/or N-, 2-O,6-O, or 3-O sulfation. Alternatively, any of the polysaccharidesdescribed above can be present or provided as a homogeneous compositioncomprised of polysaccharides having identical chain lengths, molecularweights, N-acetylation, and/or N-, 2-O, 6-O, or 3-O sulfation.

In various embodiments, heparosan-based polysaccharides that can be usedas sulfo group acceptors in any of the sulfotransfer reactions describedherein can generally be any molecular weight greater than 1,000 Da,including greater than 1,000,000 Da. In various embodiments,compositions or mixtures comprising N-deacetylated heparosanpolysaccharides can preferably have a weight-average molecular weight inthe range of at least 2,000 Da and up to 20,000 Da, or at least 9,000Da, and up to 12,500 Da. In various embodiments, sulfated polysaccharideproducts of any of the reactions described herein can comprise molecularweights associated with the addition of a single sulfo group (about 80Da), and up to the addition of sulfo groups to all available N, 2-O,3-O, and/or 6-O positions, based on the molecular weight of thepolysaccharide used as the sulfo group acceptor.

In various embodiments, in any of the methods for synthesizing anN,2,3,6-HS product described herein, any reaction mixture comprising anengineered sulfotransferase enzyme and an aryl sulfate compound canfurther comprise one or more components for repopulating the arylsulfate compound. In various embodiments, the one or more components forrepopulating the aryl sulfate compound can comprise an aryl sulfatesulfotransferase (ASST) enzyme and a secondary aryl sulfate compound.According to the present invention, the engineered sulfotransferaseenzyme has minimal or no activity with the secondary aryl sulfatecompound as a sulfo group donor. The ASST enzyme from any bacteria canbe utilized, and can either be isolated from the bacteria directly orgenerated recombinantly from an expression host in vitro. In variousembodiments, the ASST enzyme can be a recombinant ASST from E. colistrain CFT073, comprising the amino acid sequence of SEQ ID NO: 55.

In one non-limiting example, a reaction mixture comprising an N,2,6-HSproduct, NCS, and an engineered 3OST enzyme comprising the amino acidsequence SEQ ID NO: 28 can further comprise an ASST enzyme and PNS, withwhich the engineered enzyme comprising the amino acid sequence SEQ IDNO: 28 is not active. Upon being formed as a product of thesulfotransfer reaction, 4-nitrocatechol can then act as a sulfo groupacceptor for a reaction between PNS and the ASST enzyme, therebyre-synthesizing NCS for subsequent reactions with the engineered 3OSTenzyme. Alternatively, the NCS utilized for the sulfotransfer reactionto form an N,2,3,6-HS product can be generated in situ by forming areaction mixture comprising the engineered 3OST enzyme, 4-nitrocatechol,PNS, and an ASST enzyme.

In various embodiments, an engineered NST enzyme utilized in any of themethods described herein can comprise any amino acid sequence so long asthe enzyme catalyzes the transfer of a sulfo group from an aryl sulfatecompound to the amine functional group of an N-unsubstituted glucosamineresidue of a heparosan-based polysaccharide, preferably N-deacetylatedheparosan. In further embodiments, the engineered NST enzymes can bemutants of natural sulfotransferases that have HS NST activity, such asthe NDST enzymes that are members of enzyme class (EC) 2.8.2.8.According to the present invention, an engineered NST or NDST enzyme cancomprise several amino acid mutations relative to the N-sulfotransferaseand/or N-deacetylase domain of a natural NDST enzyme, in order toengineer the active site to bind and react with an aryl sulfate compoundas a sulfo group donor instead of PAPS.

Engineered NST enzymes utilized in accordance with any of the methodsdescribed herein can comprise an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 33, SEQ ID NO: 34, SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, andSEQ ID NO: 40, each of which contains several amino acid mutations maderelative to highly conserved regions within the N-sulfotransferasedomain of natural NDST enzymes within EC 2.8.2.8. In variousembodiments, engineered NST enzymes utilized in accordance with any ofthe methods described herein can also comprise an amino acid sequencehaving one or more amino residue differences or mutations from, and/oris a biological functional equivalent of, an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 33, SEQ IDNO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQID NO: 39, and SEQ ID NO: 40. Non-limiting examples of such residuedifferences include amino acid insertions, deletions, substitutions, orany combination of such changes.

In various embodiments, any of the engineered NST enzymes describedabove can further include an N-deacetylase domain that is eitheridentical or mutated relative to the N-deacetylase domain that ispresent in any of the natural NDST enzymes within EC 2.8.2.8. In variousembodiments, any of the engineered NST enzymes can further include otherdomains or fusions with other proteins to facilitate solubility orsecondary biochemical reactions.

In various embodiments, any natural NDST enzyme within EC 2.8.2.8 can beutilized to catalyze N-sulfation during the synthesis of HS products,particularly heparin products, in which engineered sulfotransferaseenzymes are utilized to catalyze the 2-O, 6-O, and/or 3-O sulfation ofthe polysaccharide. A natural NST enzyme can either include both anN-deacetylase domain and an N-sulfotransferase domain, a singleN-sulfotransferase domain, or a biologically-active fragment thereof.Reaction mixtures comprising a natural NST enzyme also comprise PAPS asa sulfo group donor.

Glucosamine residues within the heparosan-based polysaccharide that donot receive the sulfo group can be N-, 3-O, and/or 6-O sulfated,N-acetylated, or N-unsubstituted, and hexuronic acid residues caninclude GlcA or IdoA, either of which can be sulfated at the 2-Oposition. Preferably, and according to the present invention, theheparosan-based polysaccharide is N-deacetylated heparosan. In variousembodiments, the 6-O group of an N-unsubstituted glucosamine residue canalready be sulfated prior to the N-sulfation reaction.

One non-limiting example of a disaccharide unit within a heparosan-basedpolysaccharide that can react as a sulfo group acceptor with a naturalor engineered NST enzyme can comprise the structure of Formula II,below:

wherein n is an integer and R is selected from the group consisting of ahydrogen atom or a sulfo group. In various embodiments, both R groupswithin a disaccharide unit, and preferably all disaccharide units, canbe hydrogen atoms. When the sulfo acceptor polysaccharide comprises thestructure of Formula II, upon transfer of the sulfo group from an arylsulfate compound, the sulfated polysaccharide product comprises thestructure of Formula III, below:

wherein n is an integer and R is selected from the group consisting of ahydrogen atom or a sulfo group. In various embodiments, both R groupswithin the disaccharide units are hydrogen atoms.

In various embodiments, an engineered 2OST enzyme utilized in any of themethods described herein can comprise any amino acid sequence so long asthe enzyme catalyzes the transfer of a sulfo group from an aryl sulfatecompound to the 2-O position of a hexuronic acid residue within aheparosan-based polysaccharide, particularly N-sulfated HSpolysaccharides. In further embodiments, the engineered 2OST enzyme canbe a mutant of any natural 2OST enzyme, particularly an enzyme that is amember of enzyme class EC 2.8.2.-. According to the present invention,an engineered 2OST enzyme can comprise several amino acid mutationsrelative to one or more of the natural 2OST enzymes, in order toengineer the active site to bind and react with an aryl sulfate compoundas a sulfo group donor instead of PAPS.

As a non-limiting example, engineered 2OST enzymes utilized inaccordance with any of the methods described herein can comprise anamino acid sequence selected from the group consisting of SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 41, and SEQ ID NO: 42, each of which containsseveral amino acid mutations made relative to highly conserved regionswithin natural 2OST enzymes within EC 2.8.2.-. In various embodiments,engineered 2OST enzymes utilized in accordance with any of the methodsdescribed herein can also comprise an amino acid sequence having one ormore amino residue differences or mutations from, and/or is a biologicalfunctional equivalent of, an amino acid sequence selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 41, and SEQ IDNO: 42. Non-limiting examples of such residue differences include aminoacid insertions, deletions, substitutions, or any combination of suchchanges.

In various embodiments, any natural 2OST enzyme, or abiologically-active fragment thereof, can be utilized to catalyze 2-Osulfation during the synthesis of HS products, particularly N,2,3,6-HSproducts, in which engineered sulfotransferase enzymes are utilized tocatalyze the N-, 6-O, and/or 3-O sulfation of the polysaccharide.Reaction mixtures comprising a natural 2OST enzyme also comprise PAPS asa sulfo group donor.

In various embodiments, a hexuronic acid residue that can receive asulfo group from a natural or engineered 2OST enzyme can be eitherglucuronic acid or iduronic acid, and preferably iduronic acid, whileother hexuronic acid residues within the polysaccharide can beglucuronic acid or iduronic acid, either of which can be 2-O sulfated.Both glucosamine residues adjacent to the hexuronic acid residuereceiving the sulfo group can be, and preferably are, N-sulfated priorto reacting with the engineered or natural 2OST. Glucosamine residuesthat are not adjacent to the hexuronic acid residue receiving the sulfogroup can optionally be N-, 3-O, and/or 6-O sulfated, N-acetylated, orN-unsubstituted. One non-limiting example of a portion of aheparosan-based polysaccharide that can react as a sulfo group acceptorwith a natural or engineered 2OST enzyme can comprise the structure ofFormula IV, below:

When the heparosan-based polysaccharide comprises the structure ofFormula IV, the 2-O sulfated polysaccharide product comprises thestructure of Formula VI, below:

In another non-limiting example, when the hexuronic acid residue isiduronic acid, rather than glucuronic acid as illustrated in Formula IV,the heparosan-based polysaccharide comprises the structure of Formula V,below:

When the heparosan-based polysaccharide comprises the structure ofFormula V, the 2-O sulfated polysaccharide product comprises thestructure of Formula VII, below:

In various embodiments, an isolated or recombinant glucuronylC₅-epimerase enzyme can be combined in a reaction mixture along withheparosan-based polysaccharides comprising the structure of Formula IVand/or Formula V. In some embodiments, the glucuronyl C₅-epimeraseenzyme can comprise the amino acid sequence of SEQ ID NO: 29, preferablyresidues 34-617 of SEQ ID NO: 29. In other embodiments, the glucuronylC₅-epimerase enzyme can be any recombinant or natural glucuronylC₅-epimerase enzyme. In some embodiments, a glucuronyl C₅-epimeraseenzyme comprising any amino acid sequence, particularly the amino acidsequence of SEQ ID NO: 29 or residues 34-617 of SEQ ID NO: 29, can beincluded within a reaction mixture comprising N-sulfated heparosan andan engineered or natural 2OST, to form an N-sulfated, 2-O sulfated HS(N,2-HS) comprising one or more disaccharide units of 2-O sulfatediduronic acid and N-sulfo glucosamine.

In various embodiments, an engineered 6OST enzyme utilized in any of themethods described herein can comprise any amino acid sequence so long asthe enzyme catalyzes the transfer of a sulfo group from an aryl sulfatecompound to the 6-O position of a glucosamine residue within aheparosan-based polysaccharide, particularly N,2-HS polysaccharidescomprising the structure of Formula VI and/or Formula VII. In furtherembodiments, the engineered 6OST enzymes can be a mutant of any natural6OST enzyme, particularly a 6OST that is a member of enzyme class EC2.8.2.-. According to the present invention, an engineered 6OST enzymecan comprise several amino acid mutations relative to one or more of thenatural 6OST enzymes, in order to engineer the active site to bind andreact with an aryl sulfate compound as a sulfo group donor instead ofPAPS.

As non-limiting examples, engineered 6OST enzymes utilized in accordancewith any of the methods described herein can comprise an amino acidsequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ IDNO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, each of which containsseveral amino acid mutations made relative to highly conserved regionswithin natural 6OST enzymes within EC 2.8.2.-. In various embodiments,engineered 6OST enzymes utilized in accordance with any of the methodsdescribed herein can also comprise an amino acid sequence having one ormore amino residue differences or mutations from, and/or is a biologicalfunctional equivalent of, an amino acid sequence selected from the groupconsisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, andSEQ ID NO: 61. Non-limiting examples of such residue differences includeamino acid insertions, deletions, substitutions, or any combination ofsuch changes. In various embodiments, the engineered 6OST enzymecomprises the amino acid sequence of SEQ ID NO: 20.

In various embodiments, any natural 6OST enzyme, or abiologically-active fragment thereof, can be utilized to catalyze 6-Osulfation during the synthesis of HS products, particularly N,2,3,6-HSproducts, in which engineered sulfotransferase enzymes are utilized tocatalyze the N-, 2-O, and/or 3-O sulfation of the polysaccharide.According to the present invention, reaction mixtures comprising anatural 6OST enzyme also comprise PAPS as a sulfo group donor.

In various embodiments, a glucosamine residue that can receive a sulfogroup from the 6OST enzyme can be N-unsubstituted, N-sulfated, and/or3-O sulfated, prior to reacting with the enzyme. Any other glucosamineresidue within the sulfo acceptor polysaccharide can be optionally beN-, 3-O, and/or 6-O sulfated, N-acetylated, or N-unsubstituted. Any ofthe hexuronic acid residues within the heparosan-based polysaccharidecan either be iduronic acid or glucuronic acid, and can optionally be2-O sulfated, prior to reacting with the 6OST enzyme. In variousembodiments, the glucosamine residue receiving the sulfo group at the6-O position is N-sulfated, and is adjacent to a 2-O sulfated iduronicacid residue, at either or both of the non-reducing and reducing ends ofthe glucosamine residue. One non-limiting example of a portion of aheparosan-based polysaccharide that can react with a natural orengineered 6OST enzyme can comprise the structure of Formula VIII,below:

wherein X comprises any of the hexuronic acid residues depicted inFormula VIII, above.

When the heparosan-based polysaccharide comprises the structure ofFormula VIII, the 6-O-sulfated polysaccharide product comprises thestructure of Formula IX, below:

wherein X comprises any of the hexuronic acid residues depicted inFormula IX, above.

In various embodiments, an HS polysaccharide comprising the structure ofFormula VIII can be an N,2-HS polysaccharide comprising no 6-O or 3-Osulfated glucosamine residues, which upon reacting with a 6OST, forms anN-sulfated, 2-O sulfated, 6-O sulfated HS (N,2,6-HS) product. In someembodiments, N,2-HS polysaccharides produced as products of a 2OSTreaction can be isolated and purified prior to reacting with the 6OST ina separate reaction mixture, to ensure that 2-O sulfation occurs priorto 6-O sulfation. In other embodiments, 2-O sulfation of hexuronic acidresidues and 6-O sulfation of glucosamine residues can take place in thesame reaction mixture.

In various embodiments, an engineered 3OST enzyme utilized in any of themethods described herein can comprise any amino acid sequence so long asthe enzyme catalyzes the transfer of a sulfo group from an aryl sulfatecompound to the 3-O position of a glucosamine residue within aheparosan-based polysaccharide, particularly N,2-HS, N,2,6-HSpolysaccharides, and/or HS polysaccharides comprising the structure ofFormula IX. In further embodiments, engineered 3OST enzymes can be amutant of a natural 3OST enzyme, particularly a 3OST enzyme that is amember of enzyme class EC 2.8.2.23. According to the present invention,an engineered 3OST enzyme can comprise several amino acid mutationsrelative to one or more of the natural 3OST enzymes, in order toengineer the active site to bind and react with an aryl sulfate compoundas a sulfo group donor instead of PAPS.

As non-limiting examples, engineered 3OST enzymes utilized in accordancewith any of the methods described herein can comprise an amino acidsequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ IDNO: 54, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, each of whichcontains several amino acid mutations made relative to highly conservedregions within natural 3OST enzymes within EC 2.8.2.23. In variousembodiments, engineered 3OST enzymes utilized in accordance with any ofthe methods described herein can also comprise an amino acid sequencehaving one or more amino residue differences or mutations from, and/oris a biological functional equivalent of, an amino acid sequenceselected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 26, SEQID NO: 28, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58. Non-limiting examplesof such residue differences include amino acid insertions, deletions,substitutions, or any combination of such changes. In variousembodiments, the engineered 3OST enzyme comprises the amino acidsequence of SEQ ID NO: 28.

In various embodiments, any natural 3OST enzyme, or abiologically-active fragment thereof, can be utilized to catalyze 3-Osulfation during the synthesis of HS products, particularly N,2,3,6-HSproducts, in which engineered sulfotransferase enzymes are utilized tocatalyze the N-, 2-O, and/or 6-O sulfation of the polysaccharide. Invarious embodiments, reaction mixtures comprising a natural 3OST enzymealso comprise PAPS. In various embodiments, an engineered 3OST enzyme isutilized to catalyze 3-O sulfation in the synthesis of an N,2,3,6-HSproduct, even if a natural HS sulfotransferase is utilized in one ormore of the N-, 2-O, or 6-O sulfation steps.

In various embodiments, glucosamine residues within the HSpolysaccharide that can receive a sulfo group at the 3-O position areN-sulfated, and can optionally comprise a 6-O sulfo group as well. Anyother glucosamine residue within the sulfo acceptor polysaccharide canbe optionally be N-, 3-O, and/or 6-O sulfated, N-acetylated, orN-unsubstituted. In various embodiments, one or more of the glucosamineresidues within the HS polysaccharide, including the glucosamine residuebeing 3-O sulfated, can be both N-sulfated and 6-O sulfated. Accordingto the present invention, the glucosamine residue being 3-O sulfated isadjacent to an unsulfated glucuronic acid residue at the non-reducingend and an iduronic acid residue, which can optionally be 2-O sulfated,at the reducing end. Any of the other hexuronic acid residues within theHS polysaccharide can optionally be iduronic acid or glucuronic acid,and can optionally be 2-O sulfated. One non-limiting example of aportion of an HS polysaccharide that can react as a sulfo group acceptorwith a natural or engineered 3OST enzyme can comprise the structure ofFormula X, below:

wherein X is either a sulfo group or an acetate group and Y is either asulfo group or a hydroxyl group. According to the present invention, Xcan be a sulfo group and Y can be a sulfo group. When the HSpolysaccharide comprises the structure of Formula X, the 3-O sulfatedpolysaccharide product comprises the structure of Formula I, below:

wherein X is either a sulfo group or an acetate group and Y is either asulfo group or a hydroxyl group. In various embodiments, X can be asulfo group and Y can be a sulfo group.

In various embodiments, HS products synthesized by any of the methodsdescribed herein can contain one, two, three, or four sulfo groupswithin each disaccharide unit, wherein each disaccharide unit comprisesa hexuronic acid residue (GlcA or IdoA) and a glucosamine residue. In anon-limiting example, at least 45%, up to 90%, and preferably in therange of 65% to 80%, of the disaccharide units contain glucosamineresidues that are both N-sulfated and 6-O sulfated. In variousembodiments, at least 1%, up to at least 8%, and preferably in the rangeof 4% to 5%, of the glucosamine residues that are both N-sulfated and6-O sulfated are also 3-O sulfated. In another non-limiting example, atleast 1%, up to 30%, and preferably 3%, of the disaccharide units withinthe sulfated polysaccharide product comprise 2-O sulfated iduronic acidand N-sulfoglucosamine.

In various embodiments, N,2,3,6-HS polysaccharides produced by any ofthe methods of the present invention can have anticoagulant activity,including, but not limited to, the ability to bind and activateantithrombin. According to the present invention, and useful incombination with any one or more of the above aspects and embodiments,the synthesized N,2,3,6-HS product composition can comprise one or morepolysaccharides having the pentasaccharide sequence of Formula I. Insome embodiments, the N,2,3,6-HS product composition synthesizedaccording to any of the methods of the present invention can have asubstantially equivalent composition and/or anticoagulant activityrelative to heparin extracted from animal sources, including but notlimited to heparin extracted from porcine and bovine sources. In someembodiments, the N,2,3,6-HS product composition synthesized according toany of the methods of the present invention can have a substantiallyequivalent composition, physical properties, molecular weight profiles,anticoagulant activity, and/or purity relative to any of the heparinproducts described in the United States Pharmacopeia (USP), includingbut not limited to API heparin, Chemical Abstracts Service (CAS)reference standard numbers 9005-49-6 or 9041-08-1.

In some embodiments, the N,2,3,6-HS product composition synthesizedaccording to any of the methods of the present invention can beengineered to have an identical composition, physical properties,molecular weight, and anticoagulant activity relative to API heparin,while also being substantially free, or completely free, of a sulfatedpolysaccharide impurity selected from the group consisting of dermatansulfate, chondroitin sulfate, and keratan sulfate, includingcombinations thereof. Without being limited by a particular theory, itis believed that such product compositions can obviate the harmful sideeffects arising from the presence of dermatan sulfate and chondroitinsulfate found within animal-sourced heparins.

In various embodiments, antithrombin activation can be quantified as afunction of its subsequent effect on the activity of Factor IIa andFactor Xa, in terms of International Units of activity per milligram (IUmg⁻¹). In various embodiments, N,2,3,6-HS product compositions made bymethods of the present invention can have an anti-Factor IIa (anti-IIa)activity of at least about 1 IU mg⁻¹, and up to about 500 IU mg⁻¹, forexample, at least 180 IU mg⁻¹. In various embodiments, N,2,3,6-HSproduct compositions synthesized by methods of the present invention canhave an anti-Factor Xa (anti-Xa) activity of at least about 1 IU mg⁻¹,and up to about 500 IU mg⁻¹, for example, at least 180 IU mg⁻¹. Invarious embodiments, synthesized N,2,3,6-HS product compositions havingan anti-Factor Xa and/or anti-Factor IIa activity greater than 180 IUmg⁻¹ can be diluted until an activity of 180 IU mg⁻¹ is reached. Invarious embodiments, the anticoagulant activity of N,2,3,6-HS productcompositions synthesized by any of the methods of the present inventioncan be expressed as a ratio of anti-Xa activity to anti-IIa activity,ranging from at least 0.5:1, and up to at least 100:1, for example from0.9:1 to 1.1:1.

In various embodiments, polysaccharides within N,2,3,6-HS productcompositions produced by any of the methods above can have an averagemolecular weight of at least 1,500 Da, depending on the weight averagemolecular weight of polysaccharides utilized as sulfo group acceptors.In various embodiments, anticoagulant N,2,3,6-HS product mixtures canhave a weight-average molecular weight in the range of 2,000 Da to24,000 Da.

Generally, the average molecular weight of polysaccharides utilized assulfo group acceptors, particularly the average molecular weight ofN-deacetylated heparosan, can influence the average molecular weight ofN,2,3,6-HS products produced by any of the methods described herein. Invarious embodiments, the reaction time for depolymerizing andN-deacetylating heparosan can be controlled to form N-deacetylatedheparosan compositions of any desired weight-average molecular weight,such as, by non-limiting examples, at least 1,000 Da, at least 1,500 Da,at least 2,000 Da, at least 3,000 Da, at least 4,000 Da, at least 5,000Da, at least 6,000 Da, at least 7,000 Da, at least 8,000 Da, at least9,000 Da, at least 10,000 Da, at least 15,000 Da, or at least 20,000 Da.

In some embodiments, the reaction time for depolymerizing andN-deacetylating heparosan is controlled to form an N-deacetylatedheparosan composition having a weight-average molecular weight of atleast 9,000 Da, and up to 12,500 Da, such that the resulting N,2,3,6-HSproduct composition has a weight-average molecular weight of at least15,000 Da, and up to 19,000 Da. In various embodiments, less than orequal to 20% of the polysaccharide chains within the N,2,3,6-HS productcan have a molecular weight greater than 24,000 Da. In variousembodiments, and useful in combination with any one or more of the aboveaspects and embodiments, the number of polysaccharide chains within theN,2,3,6-HS product having a molecular weight between 8,000 Da and 16,000Da can be greater than the number of polysaccharide chains having amolecular weight between 16,000 Da and 24,000 Da.

In various embodiments, anticoagulant N,2,3,6-HS product compositionsproduced by any of the methods described herein can have a molecularweight profile such that: (a) the weight-average molecular weight of theanticoagulant N,2,3,6-HS product mixture is at least 15,000 Da, and upto 19,000 Da; (b) less than or equal to 20% of the polysaccharideswithin the anticoagulant N,2,3,6-HS product mixture has a molecularweight greater than 24,000 Da; and (c) the number of polysaccharidechains within the anticoagulant N,2,3,6-HS product mixture having amolecular weight between 8,000 Da and 16,000 Da is greater than thenumber of polysaccharide chains having a molecular weight between 16,000Da and 24,000 Da. In various embodiments, anticoagulant N,2,3,6-HSproduct mixtures having the molecular weight profile described above canalso have a ratio of anti-Xa activity to anti-IIa activity of at least0.9:1, up to 1.1:1, and preferably 1:1. In various embodiments,anticoagulant N,2,3,6-HS product mixtures can be prepared as a salt,particularly, as a non-limiting example, a sodium salt. In variousembodiments, the anticoagulant N,2,3,6-HS product composition can besubstantially equivalent to API heparin. In various embodiments, ananticoagulant N,2,3,6-HS product composition that is otherwisesubstantially equivalent to API heparin can be synthesized withoutcontaining any chondroitin sulfate or dermatan sulfate.

In various embodiments, engineered sulfotransferase enzymes havingbiological activity with aryl sulfate compounds as sulfo group donorscan be expressed from a nucleic acid comprising a nucleotide sequencethat encodes for any of the amino acid sequences described above.Non-limiting examples of such nucleotide sequences include SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27. Persons skilled inthe art can determine appropriate nucleotide sequences that encode forpolypeptides having the amino acid sequence of SEQ ID NOs: 33-54 and56-61, based on the nucleotide sequences above.

In various embodiments, a nucleic acid comprising any nucleotidesequence encoding for any of the engineered sulfotransferase enzymesdescribed above can be inserted into an expression vector that isengineered to be inserted into biological host cells configured toretain the expression vector and overexpress the desired enzyme.According to the present invention, the nucleic acid inserted into anexpression vector can comprise a nucleotide sequence that encodes forany of the amino acid sequences described above. According to thepresent invention, the nucleic acid inserted into an expression vectorcan comprise any of the nucleotide sequences described above.

In various embodiments, the expression vector can optionally furthercomprise one or more nucleic acid sequences or genes encoding forproteins or host recognition sites that supplement the production ofengineered sulfotransferase enzymes of the present invention.Non-limiting examples include promoter sequences, antibiotic resistancegenes, and genes encoding for fusion proteins that assist in the foldingand stability of the engineered sulfotransferase enzyme. In variousembodiments, an expression vector can further comprise the malE genefrom Escherichia coli, which encodes for maltose binding protein (MBP).For example, an expression vector can comprise the malE gene and any ofthe nucleotide sequences, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,or SEQ ID NO: 27, or any nucleotide sequence that encodes forpolypeptides having the amino acid sequence of SEQ ID NOs: 33-54 and56-61. Protein expression from those vectors can generate engineeredsulfotransferase enzymes that are fused with MBP.

Expression vectors are typically transformed into host cells from whichthe enzyme can be overexpressed and extracted. In various embodiments,host cells can be transformed with any of the expression vectorsdescribed above, non-limiting examples of which include expressionvectors comprising a nucleic acid sequence set forth in SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or any sequence thatencodes for an enzyme having the amino acid sequence of SEQ ID NOs:33-54 and 56-61. In various embodiments, the transformed host cells canbe bacterial, yeast, insect, or mammalian cells. In various embodiments,the host cells can be bacterial cells. In various embodiments, thebacterial cells can be from a non-pathogenic strain of Escherichia coli(E. coli). In various embodiments, the host cells can be yeast cells.

In various embodiments, sulfotransferase reactions within any of themethods described above can be carried out by engineered enzymescomprising at least a functional fragment of any amino acid sequencesdescribed above. In various embodiments, the invention providessubstantially pure protein purifications of engineered sulfotransferaseenzymes comprising any of the amino acid sequences above, includingfunctional fragments thereof.

In another aspect of the invention, once an HS product composition,particularly an anticoagulant N,2,3,6-HS product composition, is formedby any of the methods described above, it can be combined with aglycosaminoglycan (GAG) composition comprising at least one GAG selectedfrom the group consisting of dermatan sulfate and chondroitin sulfate,to form an HS-GAG mixture. For example, an HS-GAG mixture can compriseat least 10%, and up to 90%, of an anticoagulant N,2,3,6-HS productcomposition synthesized by any of the methods of the present invention,with the remainder comprising dermatan sulfate and/or chondroitinsulfate. In some embodiments, dermatan sulfate and/or chondroitinsulfate are added to an anticoagulant N,2,3,6-HS product compositionthat does not otherwise contain dermatan sulfate or chondroitin sulfate.In some embodiments, the anticoagulant activity of the anticoagulantN,2,3,6-HS product composition can be maintained upon the formation ofthe HS-GAG mixture.

In various embodiments, the sulfate to carboxyl ratio can describe theaverage relative abundance of sulfo groups compared to the relativeabundance of carboxyl groups within disaccharide units that comprise theanticoagulant N,2,3,6-HS product.

In one non-limiting example, an HS-GAG mixture can be formed to compriseN,2,3,6-HS products synthesized by any of the methods of the presentinvention, wherein: (a) dermatan sulfate comprises 20% of thepolysaccharides within the HS-GAG mixture; (b) the weight-averagemolecular weight of the anticoagulant N,2,3,6-HS product within theHS-GAG mixture is in the range of 7,000 Da to 8,000 Da; and (c) theanticoagulant N,2,3,6-HS product composition comprises a sulfate tocarboxyl group ratio in the range of 2.0:1 to 2.2:1. In a furtherembodiment, the HS-GAG mixture can comprise a substantially equivalentcomposition, weight-average molecular weight, and/or anticoagulantactivity relative to sulodexide.

In another non-limiting example, an HS-GAG mixture can be formed tocomprise N,2,3,6-HS products synthesized by any of the methods of thepresent invention, wherein: (a) dermatan sulfate comprises at least 10%,up to 15%, and preferably 12%, of the polysaccharides within the HS-GAGmixture; (b) chondroitin sulfate comprises at least 3%, up to 5%, andpreferably 4%, of the polysaccharides within the HS-GAG mixture; (c) theweight-average molecular weight of all of the polysaccharides within theHS-GAG mixture is in the range of 4,000 Da to 7,000 Da, and preferablyin the range of 5,000 Da to 6,000 Da; and (d) the anticoagulantN,2,3,6-HS product comprises a sulfate to carboxyl group ratio in therange of 2.0:1 to 2.2:1. In a further embodiment, the HS-GAG mixture cancomprise a substantially equivalent composition, weight-averagemolecular weight, and/or anticoagulant activity relative to danaparoid.

In another aspect of the invention, any of the N,2,3,6-HS productcompositions produced by any of the methods described herein can befurther modified to form a secondary low-molecular-weight heparinsulfate (LMW-HS) product composition upon depolymerizing and/ormodifying polysaccharides within the N,2,3,6-HS product composition.Processes for depolymerizing heparin compositions, includingunfractionated heparin compositions and API heparin compositions, arewell known in the art, and in some embodiments, any such process can beapplied to N,2,3,6-HS product compositions synthesized by any of themethods described herein. Non-limiting examples of LMW-HS productcompositions produced from synthesized N,2,3,6-HS product compositionsusing such processes, are provided in further detail, below.

In various embodiments, LMW-HS product compositions synthesized from anyof the N,2,3,6-HS products described herein can have either anequivalent or modified anticoagulant activity relative to the unmodifiedN,2,3,6-HS product. In various embodiments, a secondary LMW-HS productcomposition can have a substantially equivalent anticoagulant activityrelative to any low molecular weight heparin (LMWH) composition known inthe art. In various embodiments, LMW-HS product compositions produced byany of the methods described herein can have a ratio of anti-Xa toanti-IIa activity ranging from at least 0.5:1, up to at least 100:1,including as non-limiting examples, a ratio from at least 1.5:1, and upto at least 10:1, or a ratio from at least 20:1, and up to at least100:1.

In various embodiments, an N,2,3,6-HS product produced by any methoddescribed above can be referred to as an “unfractionated” N,2,3,6-HSproduct, relative to an LMW-HS product that is produced from theN,2,3,6-HS product.

Generally, methods of the present invention for synthesizing an LMW-HSproduct can comprise the following steps: (a) synthesizing an N,2,3,6-HSproduct according to any of the methods described herein; (b) providingone or more depolymerization agents; and (c) treating the N,2,3,6-HSproduct with the one or more depolymerization agents for a timesufficient to depolymerize at least a portion of the polysaccharideswithin the N,2,3,6-HS product, thereby forming the LMW-HS product. Invarious embodiments, the weight-average molecular weight of the LMW-HSproduct is at least 2,000 Da, and up to 12,000 Da, and is preferably inthe range of 3,000 Da to 8,000 Da.

In various embodiments, the one or more depolymerization agents can beformed by, and/or be comprised of, one or more reaction componentswithin one or more reaction mixtures, that can be combined with anunfractionated N,2,3,6-HS product to chemically and/or enzymaticallydepolymerize the N,2,3,6-HS product and form the LMW-HS product. Invarious embodiments, the selection of the depolymerization agent candetermine which chemical or enzymatic depolymerization process occurs,as well as the chemical structure and/or anticoagulant activity of theLMW-HS product that is formed as a result of the depolymerization. Suchdepolymerization processes can include, but are not limited to: chemicaland/or enzymatic β-elimination reactions; deamination reactions; andoxidation reactions, including combinations thereof. In variousembodiments, an unfractionated N,2,3,6-HS product can be treated withany combination of depolymerization agents in order to form an LMW-HSproduct.

In various embodiments, the amount of time that an unfractionatedN,2,3,6-HS product is treated with the one or more depolymerizationagents can be controlled to form an LMW-HS product with a desiredmolecular weight, chemical structure, and/or anticoagulant activity.According to the present invention, with respect to the samedepolymerization agent, the amount of time that an unfractionatedN,2,3,6-HS product is treated with the depolymerization agent can bevaried to form LMW-HS products with similar chemical structures, butdifferent molecular weights and anticoagulant activities relative toeach other.

In one-non-limiting example, an enzymatic β-elimination reaction can beperformed on the unfractionated N,2,3,6-HS product to form anenzymatically-depolymerized LMW-HS product. In various embodiments, thedepolymerization agent can comprise a carbon-oxygen lyase reactionmixture comprising at least one carbon-oxygen lyase enzyme, preferablyat least one carbon-oxygen lyase enzyme comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO:31, and SEQ ID NO: 32. In various embodiments, the unfractionatedN,2,3,6-HS product can be treated with the carbon-oxygen lyase reactionmixture for a time sufficient to catalyze β-eliminative cleavage of theunfractionated N,2,3,6-HS product and form anenzymatically-depolymerized LMW-HS product. In various embodiments, theweight-average molecular weight of the enzymatically-depolymerizedLMW-HS product can be in the range of 2,000 Da to 10,000 Da, preferably5,500 Da to 7,500 Da, and more preferably 6,500 Da. In variousembodiments, the enzymatically-depolymerized LMW-HS product can haveanticoagulant activity, particularly an anti-Xa activity in a range fromat least 70 IU mg⁻¹ and up to 120 IU mg⁻¹, and a ratio of anti-Xaactivity to anti-IIa activity in the range of 1.5:1 to 2.5:1. In variousembodiments, the enzymatically-depolymerized LMW-HS product can comprisepolysaccharides having a 4,5-unsaturated uronic acid residue at thenon-reducing end. In various embodiments, theenzymatically-depolymerized LMW-HS product can comprise a substantiallyequivalent chemical structure, weight-average molecular weight, and/oranticoagulant activity relative to tinzaparin.

In another non-limiting example, a chemical β-elimination reaction canbe performed on the unfractionated N,2,3,6-HS product to form achemically β-eliminative, LMW-HS product. In various embodiments, thedepolymerization agent for a chemical β-elimination reaction cancomprise a base, preferably a base selected from the group consisting ofsodium hydroxide, a quaternary ammonium hydroxide, and a phosphazenebase, including any combination thereof, and the unfractionatedN,2,3,6-HS product can be treated with the base for a time sufficient tocause β-eliminative cleavage of the unfractionated N,2,3,6-HS productand form a chemically β-eliminative, LMW-HS product.

In various embodiments, the step of treating the unfractionatedN,2,3,6-HS product with the depolymerization agent can comprise thefollowing sub-steps: (i) reacting the unfractionated N,2,3,6-HS productwith a benzethonium salt, preferably benzethonium chloride, to form abenzethonium HS salt; and (ii) combining the benzethonium HS salt with areaction mixture comprising the base for a time sufficient to form thechemically β-eliminative, LMW-HS product. In various embodiments, theweight-average molecular weight of the chemically β-eliminative, LMW-HSproduct can be at least 2,000 Da, up to 10,000 Da, and preferably in therange of 2,000 Da to 6,000 Da. In various embodiments, the chemicallyβ-eliminative, LMW-HS product can comprise polysaccharides having a4,5-unsaturated uronic acid residue at the non-reducing end. Accordingto the present invention, and useful in combination with any one or moreof the above aspects and embodiments, the chemically (3-eliminative,LMW-HS product can have anticoagulant activity, particularly an anti-Xaactivity in a range from 80 IU mg⁻¹ up to 160 IU mg⁻¹, an anti-IIaactivity in a range from 2 IU mg⁻¹ up to 40 IU mg⁻¹, and/or a ratio ofanti-Xa activity to anti-IIa activity in the range of 3.0:1 to 100:1.

In various embodiments, once the benzethonium HS salt is formed, it canbe subsequently treated with a base for a time sufficient to form thechemically β-eliminative, LMW-HS product. In various embodiments, thebase can be a quaternary ammonium hydroxide, preferably benzyltrimethylammonium hydroxide (Triton® B). In various embodiments, theweight-average molecular weight of the chemically β-eliminative, LMW-HSproduct can be in the range of 3,000 Da to 4,200 Da, and preferably3,600 Da. In various embodiments, the anti-Xa activity of the chemicallyβ-eliminative, LMW-HS product can be in a range from at least 80 IU mg⁻¹and up to 120 IU mg⁻¹, the anti-IIa activity can be in a range from atleast 5 IU mg⁻¹ and up to 20 IU mg⁻¹, and/or the ratio of anti-Xaactivity to anti-IIa activity of the chemically β-eliminative, LMW-HSproduct can be in the range of 8.0:1 to 10.0:1. In various embodiments,LMW-HS product can comprise a substantially equivalent chemicalstructure, weight-average molecular weight, and/or anticoagulantactivity relative to bemiparin.

In various embodiments, the benzethonium HS salt can instead be furthermodified prior to reacting with the base. In one non-limiting example,the benzethonium HS salt can be converted to a benzyl ester form of HSupon reacting with a benzyl halide, particularly benzyl chloride. Invarious embodiments, the conversion to the benzyl ester can take placewithin a chlorinated solvent, including but not limited to methylenechloride and chloroform.

In various embodiments, once the benzyl ester HS is formed, it can besubsequently reacted with a base to initiate depolymerization. Invarious embodiments, the base can be sodium hydroxide. In variousembodiments, the chemically β-eliminative, LMW-HS product can comprisepolysaccharides having a 1,6-anhydromannose or 1,6-anhydroglucosamineresidue at the reducing end in addition to the 4,5-unsaturated uronicacid residue at the non-reducing end. In various embodiments, theweight-average molecular weight of the chemically β-eliminative, LMW-HSproduct can be in the range of 3,800 Da to 5,000 Da, preferably 4,500Da. In various embodiments, the anti-Xa activity of the chemicallyβ-eliminative, LMW-HS product can be in a range from at least 90 IU mg⁻¹and up to 125 IU mg⁻¹, the anti-IIa activity can be in a range from atleast 20 IU mg⁻¹ and up to 35 IU mg⁻¹, and/or the ratio of anti-Xaactivity to anti-IIa activity of the chemically β-eliminative, LMW-HSproduct can be in the range of 3.3:1 to 5.3:1. In various embodiments,the chemically β-eliminative, LMW-HS product can comprise asubstantially equivalent chemical structure, weight-average molecularweight, and/or anticoagulant activity relative to enoxaparin.

In various embodiments, the benzyl ester HS can instead be transalifiedin the presence of a benzethonium salt, preferably benzethoniumchloride, in order to form a benzethonium benzyl ester HS, which canthen subsequently de-polymerized using a base. In various embodiments,the base is a phosphazene base, preferably2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diaza-phosphorine(BEMP). After depolymerization is complete, the remaining benzyl esterswithin the chemically β-eliminative, LMW-HS product can be saponifiedand removed. In various embodiments, the weight-average molecular weightof the chemically β-eliminative, LMW-HS product can be in the range of2,000 Da to 3,000 Da, and is preferably 2,400 Da. In variousembodiments, the anti-Xa activity of the chemically β-eliminative,LMW-HS product can less than or equal to 160 IU mg⁻¹, and/or the ratioof anti-Xa activity to anti-IIa activity can be at least 20:1, up to100:1, and preferably 80:1. In various embodiments, the chemicallyβ-eliminative, LMW-HS product can comprise a substantially equivalentchemical structure, weight-average molecular weight, and/oranticoagulant activity relative to semuloparin.

In various embodiments, unfractionated N,2,3,6-HS products canoptionally be depolymerized by both an enzymatic and a chemicalβ-elimination reaction. For example, an enzymatically-depolymerizedLMW-HS product can subsequently be subjected to a chemical β-eliminationreaction by reacting with a base. In another example, a chemicallyβ-eliminative, LMW-HS product can subsequently be subjected to anenzymatic β-elimination reaction by reacting one or more carbon-oxygenlyase enzymes.

In another non-limiting example, deamination reaction can be performedon the unfractionated N,2,3,6-HS product to form a deaminated LMW-HSproduct. In various embodiments, the depolymerization agent can comprisea deamination reaction mixture comprising a deamination agent,preferably a deamination agent selected from the group consisting ofisoamyl nitrate and nitrous acid, for a time sufficient to causedeaminative cleavage of the unfractionated N,2,3,6-HS product, therebyforming a deaminated LMW-HS product.

In various embodiments, the deamination agent can be nitrous acid. Invarious embodiments, the deamination reaction mixture can comprisestoichiometric quantities of an acid, preferably acetic acid orhydrochloric acid, and an alkali or alkaline earth metal nitrite salt,preferably sodium nitrite, to form nitrous acid in situ. In variousembodiments, the deaminated LMW-HS product can comprise polysaccharideshaving a 2,5-anhydro-D-mannose residue at the reducing end. In variousembodiments, the weight-average molecular weight of the deaminatedLMW-HS product can be in the range of 2,000 Da to 10,000 Da, preferablyin the range of 4,000 Da to 6,000 Da. According to the presentinvention, and useful in combination with any one or more of the aboveaspects and embodiments, the deaminated LMW-HS product can haveanticoagulant activity, particularly having a ratio of anti-Xa activityto anti-IIa activity in the range of 2.0:1 to 4.5:1.

In one non-limiting example, the weight-average molecular weight of thedeaminated LMW-HS product can be in the range of 3,600 Da to 5,500 Da,preferably 4,300 Da. In various embodiments, the anti-Xa activity of thedeaminated LMW-HS product can be in a range from at least 95 IU mg⁻¹ andup to not more than 130 IU mg⁻¹, and/or and the ratio of anti-Xaactivity to anti-IIa activity can be in the range of at least 2.5:1 andup to 4.0:1. In various embodiments, the deaminated LMW-HS product cancomprise a substantially equivalent chemical structure, weight-averagemolecular weight, and/or anticoagulant activity relative to nadroparin.

In another non-limiting example, the weight-average molecular weight ofthe deaminated LMW-HS product can be in the range of 5,600 Da to 6,400Da, preferably 6,000 Da. In various embodiments, the anti-Xa activity ofthe deaminated LMW-HS product can be in a range from at least 110 IUmg⁻¹ and up to not more than 210 IU mg⁻¹, the anti-IIa activity can bein a range from at least 35 IU mg⁻¹ and up to not more than 100 IU mg⁻¹,and/or the ratio of anti-Xa activity to anti-IIa activity of thedeaminated LMW-HS product can be at least 1.9:1, and up to 3.2:1. Invarious embodiments, the deaminated LMW-HS product can comprise asubstantially equivalent chemical structure, weight-average molecularweight, and/or anticoagulant activity relative to dalteparin.

In another non-limiting example, the weight-average molecular weight ofthe deaminated LMW-HS product can be in the range of 4,200 Da to 4,600Da, preferably 4,400 Da, the anti-Xa activity can be in a range from atleast 98 IU mg⁻¹ and up to 155 IU mg⁻¹, and the ratio of anti-Xaactivity to anti-IIa activity of the deaminated LMW-HS product can be atleast 4.0:1, and up to 4.5:1, preferably 4.2:1. In various embodiments,the deaminated LMW-HS product can comprise a substantially equivalentchemical structure, weight-average molecular weight, and/oranticoagulant activity relative to reviparin.

In another non-limiting example, the deamination agent is isoamylnitrate, the weight-average molecular weight of the deaminated LMW-HSproduct can be in the range of 5,000 Da to 5,600 Da, preferably 5,400Da, the anti-Xa activity can be in a range from at least 80 IU mg⁻¹ andup to 120 IU mg⁻¹, and the ratio of anti-Xa activity to anti-IIaactivity of the deaminated LMW-HS product can be at least 2.0:1, and upto 2.5:1, preferably 2.4:1. In various embodiments, the deaminatedLMW-HS product can comprise a substantially equivalent chemicalstructure, weight-average molecular weight, and/or anticoagulantactivity relative to certoparin.

In another non-limiting example, an oxidation reaction can be performedon the unfractionated N,2,3,6-HS product to form an oxidized LMW-HSproduct. In various embodiments, the depolymerization agent can comprisean oxidation agent, preferably an oxidation agent selected from thegroup consisting of a peroxide or a superoxide, and more preferablyhydrogen peroxide to form an oxidized LMW-HS product. In variousembodiments, the step of treating an unfractionated N,2,3,6-HS productwith the oxidation agent can comprise the following sub-steps: (i)acidifying the unfractionated N,2,3,6-HS product to form an acidified HSproduct; (ii) combining the acidified HS product with the oxidationreaction mixture; and (iii) incubating the acidified HS product withinthe oxidation reaction mixture at a temperature of at least than 50° C.for a time sufficient to form the oxidized LMW-HS product.

In various embodiments, the sub-step of acidifying the unfractionatedN,2,3,6-HS product can comprise the addition of a reaction mixturecomprising an acid, preferably ascorbic acid, to the HS product to formthe acidified HS product. Alternatively, the sub-step of acidifying theunfractionated N,2,3,6-HS product can further comprise the sub-steps of:loading the unfractionated N,2,3,6-HS product into a cation exchangeresin, preferably a cation exchange resin suspended within achromatography column; and eluting the unfractionated N,2,3,6-HS productfrom the cation exchange resin, forming the acidified HS product. Invarious embodiments, the pH of the acidified HS product can be at least3.0, and up to 5.0, and preferably in a range of 3.0 to 3.5.

In various embodiments, the weight-average molecular weight of theoxidized LMW-HS product can be in the range of 2,000 Da to 12,000 Da,preferably in the range of 4,000 Da to 6,000 Da. In various embodiments,the oxidized LMW-HS product can have anticoagulant activity,particularly in which the ratio of anti-Xa activity to anti-IIa activityis in the range of 1.5:1 to 3.0:1.

In one non-limiting example, the weight-average molecular weight of theoxidized LMW-HS product can be in the range of at least 4,000 Da up to6,000 Da, and is preferably 5,000 Da, the anti-Xa activity of theoxidized LMW-HS product is in a range from at least 95 IU mg⁻¹ and up tonot more than 110 IU mg⁻¹, and the ratio of anti-Xa activity to anti-IIaactivity is at least 1.5:1, and up to 3.0:1. In various embodiments, theoxidized LMW-HS product can comprise a substantially equivalent chemicalstructure, pH, weight-average molecular weight, and/or anticoagulantactivity relative to parnaparin.

In another non-limiting example, the weight-average molecular weight ofthe oxidized LMW-HS product can be in the range of 5,500 Da to 6,500 Da,preferably 6,000 Da, and the ratio of anti-Xa activity to anti-IIaactivity is at least 2.0:1, and up to 2.5:1. In various embodiments, theoxidized LMW-HS product can comprise a substantially equivalent chemicalstructure, pH, weight-average molecular weight, and/or anticoagulantactivity relative to ardeparin.

In various embodiments, LMW-HS products can be formed from N,2,3,6-HSproducts synthesized by any of the methods of the present invention,wherein the N,2,3,6-HS product has a substantially equivalent molecularweight profile and anticoagulant activity relative to API heparin,namely that: (a) the weight-average molecular weight of the N,2,3,6-HSproduct is at least 15,000 Da, and up to 19,000 Da; (b) less than orequal to 20% of the polysaccharides within the N,2,3,6-HS product have amolecular weight greater than 24,000 Da; (c) the number ofpolysaccharide chains within the N,2,3,6-HS product having a molecularweight between 8,000 Da and 16,000 Da is greater than the number ofpolysaccharide chains having a molecular weight between 16,000 Da and24,000 Da; (d) the anti-Xa activity of the N,2,3,6-HS product is about180 IU mg⁻¹; (e) the anti-IIa activity of the N,2,3,6-HS product isabout 180 IU mg⁻¹; and the ratio of anti-Xa activity to anti-IIaactivity in the N,2,3,6-HS product is at least 0.9:1, and up to 1.1:1.In various embodiments, LMW-HS products can be formed from N,2,3,6-HSproducts synthesized by any of the methods of the present invention,wherein the N,2,3,6-HS product is produced from N-deacetylated heparosancompositions having a weight average molecular weight of less than 9,000Da, non-limiting examples of which are less than 8,000 Da, less than7,000 Da, less than 6,000 Da, less than 5,000 Da, less than 4,000 Da,less than 3,000 Da, or less than 2,000 Da.

In another aspect of the invention, kits for forming N,2,3,6-HS orLMW-HS products, particularly anticoagulant N,2,3,6-HS or LMW-HSproducts, according to any of the methods described above, are provided.In various embodiments, the kit can comprise at least one engineeredaryl sulfate-dependent sulfotransferase and at least one aryl sulfatecompound, preferably PNS or NCS In various embodiments, the kit cancomprise an engineered NST, an engineered 2OST, an engineered 6OST,and/or an engineered 3OST, each of which is dependent on reacting withan aryl sulfate compound as a sulfo group donor to catalyze a transferof the sulfo group to a polysaccharide, preferably a heparosan-basedpolysaccharide. In various embodiments, the kit can further comprise anyof the starting polysaccharides or sulfated polysaccharides describedabove, including heparosan and/or other HS polysaccharides. In variousembodiments, the kit can further comprise an epimerase, preferably anepimerase comprising the amino acid sequence of SEQ ID NO: 29, and morepreferably an epimerase comprising amino acid residues 34-617 of SEQ IDNO: 29. In various embodiments, the kit can comprise any of thecomponents and/or reaction mixtures for chemically N-sulfatingheparosan-based polysaccharides, particularly N-deacetylated heparosan.In various embodiments, the kit can comprise any of the componentsand/or reaction mixtures for isolating and purifying heparosan from ahost, preferably a bacterial host, and more preferably E. coli. Invarious embodiments, the kit can comprise any of the components and/orreaction mixtures for depolymerizing an N,2,3,6-HS product according toany of the methods described above, in order to form any of theenzymatically-depolymerized, chemically β-eliminative, deaminated, oroxidized LMW-HS products.

According to the present invention, and useful in combination with anyone or more of the above aspects and embodiments, any of thenon-anticoagulant or anticoagulant HS products, N,2,3,6-HS products,and/or LMW-HS products prepared according to any of the methodsdescribed above can be prepared as pharmaceutically-acceptable salts,particularly alkali or alkali earth salts including, but not limited to,sodium, lithium, or calcium salts.

These and other embodiments of the present invention will be apparent toone of ordinary skill in the art from the following detaileddescription.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show an example reaction mechanism between the human 3OSTenzyme, PAPS, and an N-sulfated, 6-O sulfated glucosamine residue withinheparan sulfate.

FIG. 2 shows a non-limiting example of an N-deacetylated heparosanpolysaccharide capable of reacting as a sulfo group acceptor for bothnatural NDST enzymes and engineered NST enzymes that can be used inaccordance with methods of the present invention.

FIGS. 3A-3C show a multiple sequence alignment for theN-sulfotransferase domains of fifteen natural NDST enzymes within enzymeclass EC 2.8.2.8, illustrating conserved amino acid sequence motifs thatare present regardless of overall sequence identity.

FIGS. 4A-4C show a reaction mechanism between conserved residues withinthe N-sulfotransferase domain of a natural NDST enzyme, PAPS, andN-deacetylated heparosan.

FIG. 5 shows a three-dimensional model of an aryl sulfate compound boundwithin the active site of a first group of engineered NST enzymes,superimposed over the crystal structure of PAPS bound within theN-sulfotransferase domain of a natural human NDST enzyme.

FIG. 6 shows an alternate view of the modelled active site of theengineered NST enzyme shown in FIG. 5 , illustrating amino acidmutations present within the active site.

FIG. 7 shows a three-dimensional model of an aryl sulfate compound boundwithin the active site of a second group of engineered NST enzymes,superimposed over the crystal structure of the N-sulfotransferase domainof a natural human NDST enzyme.

FIG. 8 shows an alternate view of the modelled active site of theengineered NST enzyme shown in FIG. 7 , illustrating amino acidmutations present within the active site.

FIG. 9 shows a sequence alignment of polypeptides comprising the aminoacid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, respectively, depicting the positionand identity of amino acid residues differences between each of theillustrated sequences and relative to the human NDST1 enzyme.

FIG. 10 shows the 2-O sulfation of one non-limiting example of anN-sulfated heparosan polysaccharide, catalyzed by either a natural orengineered 2OST enzyme in accordance with methods of the presentinvention, wherein the polysaccharide comprises N-sulfated,N-acetylated, and unsubstituted glucosaminyl residues.

FIG. 11 shows the 2-O sulfation of a glucuronic acid residue withinanother non-limiting example of an N-sulfated heparosan polysaccharide,catalyzed by either a natural or engineered 2OST enzyme in accordancewith methods of the present invention.

FIG. 12 shows the 2-O sulfation of an iduronic acid residue within theN-sulfated heparosan polysaccharide shown in FIG. 11 , catalyzed byeither a natural or engineered 2OST enzyme in accordance with methods ofthe present invention.

FIG. 13 shows the 2-O sulfation of a glucuronic acid residue and aniduronic acid residue within the N-sulfated heparosan polysaccharideshown in FIG. 11 , catalyzed by either a natural or engineered 2OSTenzyme in accordance with methods of the present invention.

FIGS. 14A-14D show a multiple sequence alignment for twelve natural 2OSTenzymes within EC 2.8.2.-, illustrating conserved amino acid sequencemotifs that are present regardless of overall sequence identity.

FIGS. 15A-15C show a reaction mechanism between conserved residueswithin a natural 2OST enzyme, PAPS, and a hexuronic acid residue withinN-sulfated heparosan.

FIG. 16 shows a three-dimensional model of an aryl sulfate compoundbound within the active site of an engineered 2OST enzyme, superimposedover the crystal structure of PAPS bound within the active site of thechicken 2OST enzyme.

FIG. 17 shows the 6-O sulfation of one non-limiting example of anN-sulfated, 2-O sulfated heparan sulfate polysaccharide, catalyzed byeither a natural or engineered 6OST enzyme in accordance with methods ofthe present invention, wherein multiple glucosamine residues within thepolysaccharide are capable of receiving a sulfate group.

FIGS. 18A-18C show a multiple sequence alignment for fifteen natural6OST enzymes within EC 2.8.2.-, illustrating conserved amino acidsequence motifs that are present regardless of overall sequenceidentity.

FIGS. 19A-19C show a reaction mechanism between conserved residueswithin a natural 6OST enzyme, PAPS, and an N-sulfated glucosamineresidue within heparan sulfate.

FIG. 20 shows a three-dimensional model of an aryl sulfate compoundbound within the active site of an engineered 6OST enzyme, superimposedover the crystal structure of PAPS bound within the zebrafish 6OST3enzyme.

FIG. 21 shows a sequence alignment of polypeptides comprising the aminoacid sequences of SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22,respectively, depicting the position and identity of amino acid residuesdifferences between each of the illustrated sequences and relative tothe mouse 6OST1 enzyme.

FIG. 22 shows the 3-O sulfation of one non-limiting example of anN-sulfated, 2-O sulfated, 6-O sulfated heparan sulfate polysaccharide,catalyzed by either a natural or engineered 3OST enzyme in accordancewith methods of the present invention.

FIGS. 23A-23C show a multiple sequence alignment for fifteen natural3OST enzymes within EC 2.8.2.23, illustrating conserved amino acidsequence motifs that are present regardless of overall sequenceidentity.

FIG. 24 shows a three-dimensional model of an aryl sulfate compoundbound within the active sites of three superimposed engineered 3OSTenzymes, which themselves are superimposed over the crystal structure ofPAPS bound within the mouse 3OST1 enzyme.

FIG. 25 shows a sequence alignment of polypeptides comprising the aminoacid sequences of SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28,respectively, depicting the position and identity of amino acid residuesdifferences between each of the illustrated sequences and relative tothe mouse 3OST1 enzyme.

FIG. 26 shows a series of overlaid SAX-HPLC chromatograms of digestedN-sulfated heparosan products synthesized using an engineered NSTenzyme, compared to commercial standards.

FIGS. 27A-27B show a series of LCMS chromatograms of digested N-,2-O-sulfated polysaccharide products synthesized using an engineered2OST having the amino acid sequence SEQ ID NO: 14 or SEQ ID NO: 16,respectively.

FIGS. 28A-28C shows an LCMS chromatogram of digested N-, 2-O-,6-O-sulfated polysaccharide products synthesized using an engineered6OST having the amino acid sequence SEQ ID NO: 18, SEQ ID NO: 20, or SEQID NO: 22, respectively.

FIGS. 29A-29B show a series of overlaid LCMS chromatograms of digestedN-, 2-O-, 6-O-, 3-O-sulfated polysaccharide products synthesized usingengineered 3OST enzymes having the amino acid sequence SEQ ID NO: 24,SEQ ID NO: 26, or SEQ ID NO: 28, compared to a series of disaccharideand polysaccharide standards.

FIG. 30 shows the reaction scheme for deuterium labeling of protons ofinterest for nuclear magnetic resonance (NMR) studies.

FIG. 31 shows an expanded view of ¹H-NMR spectra for engineered 3OSTenzymes having the amino acid sequence SEQ ID NO: 24, SEQ ID NO: 26, orSEQ ID NO: 28, either with PNS or NCS.

FIG. 32 shows a magnified view of the 3.5 ppm to 4.5 ppm region of the¹H-NMR spectra illustrated in FIG. 31 .

FIG. 33 shows a SAX-HPLC chromatogram of a chemically N-sulfatedpolysaccharide product, compared to a commercial standard.

FIG. 34 shows a SAX-HPLC chromatogram of an enzymatically 2-O sulfatedpolysaccharide product prepared using the chemically N-sulfatedpolysaccharide product of Example 7 as the sulfo acceptorpolysaccharide, compared to a commercial standard.

FIG. 35 shows a SAX-HPLC chromatogram of an enzymatically 2-O sulfatedpolysaccharide product prepared using the chemically N-sulfatedpolysaccharide product of Example 7 as the sulfo acceptor polysaccharideand with a C₅-hexuronyl epimerase included in the reaction mixture,compared to a commercial standard.

FIG. 36 shows a SAX-HPLC chromatogram of an enzymatically 6-O sulfatedpolysaccharide product prepared using the sulfated polysaccharideproduct of Example 8 as the sulfo group acceptor, compared to acommercial standard.

DEFINITIONS

The term, “active site,” refers to sites in catalytic proteins, in whichcatalysis occurs, and can include one or more substrate binding sites.Active sites are of significant utility in the identification ofcompounds that specifically interact with, and modulate the activity of,a particular polypeptide. The association of natural ligands orsubstrates with the active sites of their corresponding receptors orenzymes is the basis of many biological mechanisms of action. Similarly,many compounds exert their biological effects through association withthe active sites of receptors and enzymes. Such associations may occurwith all or any parts of the active site. An understanding of suchassociations helps lead to the design of engineered active sites withinsulfotransferases that are capable of binding to and reacting with arylsulfate compounds instead of PAPS.

The term, “amino acid,” refers to a molecule having the structurewherein a central carbon atom (the alpha-carbon atom) is linked to ahydrogen atom, a carboxylic acid group (the carbon atom of which isreferred to herein as a “carboxyl carbon atom”), an amino group (thenitrogen atom of which is referred to herein as an “amino nitrogenatom”), and a side chain group, R. When incorporated into a peptide,polypeptide, or protein, an amino acid loses one or more atoms of itsamino and carboxylic groups in the dehydration reaction that links oneamino acid to another. As a result, when incorporated into a protein, anamino acid is referred to as an “amino acid residue.” In the case ofnaturally occurring proteins, an amino acid residue's R groupdifferentiates the 20 amino acids from which proteins are synthesized,although one or more amino acid residues in a protein may be derivatizedor modified following incorporation into protein in biological systems(e.g., by glycosylation and/or by the formation of cysteine through theoxidation of the thiol side chains of two non-adjacent cysteine aminoacid residues, resulting in a disulfide covalent bond that frequentlyplays an important role in stabilizing the folded conformation of aprotein, etc.). Additionally, when an alpha-carbon atom has fourdifferent groups (as is the case with the 20 amino acids used bybiological systems to synthesize proteins, except for glycine, which hastwo hydrogen atoms bonded to the carbon atom), two differentenantiomeric forms of each amino acid exist, designated D and L. Inmammals, only L-amino acids are incorporated into naturally occurringpolypeptides. Engineered sulfotransferase enzymes utilized in accordancewith methods of the present invention can incorporate one or more D- andL-amino acids, or can be comprised solely of D- or L-amino acidresidues.

Non-naturally occurring amino acids can also be incorporated into any ofthe sulfotransferase enzymes utilized in accordance with the methods ofthe present invention, particularly engineered sulfotransferase enzymeshaving aryl sulfate-dependent activity. Examples of such amino acidsinclude, without limitation, alpha-amino isobutyric acid, 4-aminobutyric acid, L-amino butyric acid, 6-amino hexanoic acid, 2-aminoisobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butyl alanine, phenylglycine, cyclohexyl alanine,beta-alanine, fluoro-amino acids, designer amino acids (e.g.,beta-methyl amino acids, alpha-methyl amino acids, alpha-methyl aminoacids) and amino acid analogs in general.

The term, “and/or,” when used in the context of a listing of entities,refers to the entities being present singly or in combination. Thus, forexample, the phrase “A, B, C, and/or D” includes A, B, C, and Dindividually, but also includes any and all combinations andsub-combinations of A, B, C, and D.

The term, “API heparin,” refers to the form of heparin that is regulatedfor administering to patients, and which conforms to the United StatesPharmacopeia (USP) reference standard with respect to identity,strength, quality, purity, and potency. Properties defined by the USPmonograph for heparin sodium include: a characteristic ¹H-NMR spectrum;chromatographic purity, particularly with respect to dermatan sulfateand oversulfated chondroitin sulfate; anti-Factor Xa activity;anti-Factor IIa activity; the ratio of anti-factor Xa activity relativeto anti-factor IIa activity; the presence or absence of inorganic andinorganic impurities; and a characteristic molecular weight distributionor profile. In particular, the USP Heparin Sodium standard has ananti-Factor Xa activity of not less than 180 IU mg⁻¹; an anti-factor IIaactivity of not less than 180 IU mg⁻¹; a ratio of anti-Factor Xaactivity to anti-Factor IIa activity of 0.9-1.1; the amount ofpolysaccharide chains greater than 24,000 Da is less than 20% of aheparin sample; the amount of polysaccharide chains between 8,000 Da and16,000 Da being greater than the amount of polysaccharide chains between16,000 Da and 24,000 Da within the heparin sample; and a weight averagemolecular weight of the heparin sample in the range of at least 15,000Da and up to 19,000 Da.

The terms, “aryl sulfate” or “aryl sulfate compound,” refer to anycompound, functional group, or substituent derived from an aromatic ringin which one or more of the hydrogen atoms directly bonded to thearomatic ring is replaced by a sulfate functional group. Typically, thesulfate functional group is covalently bound to the aromatic moiety ofan aryl sulfate compound through a sulfate ester linkage. Exemplary arylsulfate compounds that can donate a sulfo group to a polysaccharide,particularly a heparosan-based polysaccharide, using any of theengineered sulfotransferases include, but are not limited to,p-nitrophenyl sulfate (PNS), 4-methylumbelliferyl sulfate (MUS),7-hydroxycoumarin sulfate, phenyl sulfate, 4-acetylphenyl sulfate,indoxyl sulfate, 1-naphthyl sulfate, 2-naphthyl sulfate, and4-nitrocatechol sulfate (NCS).

The term, “aryl sulfate-dependent sulfotransferase,” refers to thecollective group of engineered sulfotransferases that possess biologicalor catalytic activity with aryl sulfate compounds as sulfo donors.Non-limiting examples of aryl sulfate compounds upon which thebiological activity of the sulfotransferase can be dependent include PNSand NCS. As described herein, engineered sulfotransferases havingbiological activity with aryl sulfate compounds as sulfo group donorscan possess biological activity with polysaccharides, particularlyheparosan-based polysaccharides, as sulfo group acceptors. “Arylsulfate-dependent sulfotransferase” also includes both nucleic acids andpolypeptides encoding for any aryl sulfate-dependent sulfotransferase,including mutants derived from the sequences disclosed herein.

The term, “average molecular weight,” with respect to any of thepolysaccharide starting materials, intermediates, and/or products usedor generated according to any of the methods of the present invention,and unless otherwise indicated, can refer to any accepted measure ofdetermining the molar mass distribution or molar mass average of amixture of polymers having varying degrees of polymerization,functionalization, and molar mass, including but not limited to“number-average molecular weight,” “mass-average molecular weight,”“weight-average molecular weight,” “Z (centrifugation) average molarmass,” or “viscosity average molar mass.”

The term, “weight-average molecular weight,” refers to a method ofreporting the average molecular weight of polysaccharides in a mixture,calculated using the mole fraction distribution of the polysaccharideswithin the sample, using the equation

${{\overset{\_}{M}}_{w} = \frac{\Sigma_{i}N_{i}M_{i}^{2}}{\Sigma_{i}N_{i}M_{i}}},$wherein N_(i) is the number of polysaccharides of molecular mass M_(i).

The term, “number-average molecular weight,” refers to a method ofreporting the average molecular weight of polysaccharides in a mixture,calculated by dividing the total weight of all of the polysaccharides inthe sample divided by the number of polysaccharides in a sample, usingthe equation,

${{\overset{\_}{M}}_{N} = \frac{\Sigma_{i}N_{i}M_{i}}{\Sigma_{i}N_{i}}},$wherein N_(i) is the number of polysaccharides of molecular mass M_(i).Accordingly, the weight-average molecular weight, M _(w), is necessarilyskewed toward higher values corresponding to polysaccharides within thesample that are larger than other polysaccharides within the samemixture, and will always be larger than the number-average molecularweight, M _(n), except when the sample is monodisperse, and M _(w)equals M _(n). If a particular sample of polysaccharides within thesample has a large dispersion of actual weights, then M _(w) will bemuch larger than M _(n). Conversely, as the weight dispersion ofpolysaccharides in a sample narrows, M _(w) approaches M _(n).

The terms, “relative molecular weight” or “relative molar mass” (M_(r)),refers to another method of reporting the average molecular weight ofpolysaccharides in a mixture as a unitless quantity, most broadlydetermined by dividing the average mass of the molecule by an atomicmass constant, such as 1 atomic mass unit (amu) or 1 Dalton (Da). Withrespect to polysaccharides, M_(r) does not take into account thedifferent chain-lengths, functionalization, and/or weight distributionof the polysaccharides in the sample, and instead simply represents thetrue average mass of the polysaccharides in the sample in a mannersimilar to small molecules.

The terms, “biological activity” or “catalytic activity,” refer to theability of an enzyme to catalyze a particular chemical reaction byspecific recognition of a particular substrate or substrates to generatea particular product or products. In some embodiments, the engineeredenzymes of the present invention possess a biological or catalyticactivity that is dependent on binding and reacting with aryl sulfatecompounds, particularly PNS, as substrates. Additionally, someengineered enzymes are capable of having promiscuous catalytic activitywith one or more alternate aryl sulfate compounds in addition to PNS,including but not limited to MUS, 7-hydroxycoumarin sulfate, phenylsulfate, 4-acetylphenyl sulfate, indoxyl sulfate, 1-naphthyl sulfate,2-naphthyl sulfate, and NCS.

The term, “coding sequence,” refers to that portion of a nucleic acid,for example, a gene, that encodes an amino acid sequence of a protein.

The term, “codon-optimized” refers to changes in the codons of thepolynucleotide encoding a protein to those preferentially used in aparticular organism such that the encoded protein is efficientlyexpressed in the organism of interest. Although the genetic code isdegenerate in that most amino acids are represented by several codons,it is well known that codon usage by particular organisms is non-randomand biased toward particular codon triplets. In some embodiments of theinvention, the polynucleotide encoding for an engineered enzyme may becodon optimized for optimal production from the host organism selectedfor expression.

The terms, “corresponding to,” “reference to,” or “relative to,” whenused in the context of the numbering of a given amino acid orpolynucleotide sequence, refers to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. In other words, theresidue number or residue position of a given polymer is designated withrespect to the reference sequence rather than by the actual numericalposition of the residue within the given amino acid or polynucleotidesequence.

The term, “deletion,” refers to modification of a polypeptide by removalof one or more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, the net result of which isretaining the catalytic activity of the reference polypeptide. Deletionscan be directed to the internal portions and/or terminal portions of apolypeptide. Additionally, deletions can comprise continuous segments orthey can be discontinuous.

The term, “disaccharide unit,” refers to the smallest repeating backboneunit within many polysaccharides, including linear polysaccharides, inwhich the smallest repeating unit consists of two sugar residues. Withrespect to a heparosan-based polysaccharide, the disaccharide unitconsists of a hexuronic acid residue and a glucosamine residue, eitherof which can be functionalized and in which the hexuronic acid residuecan either be glucuronic acid or iduronic acid. Each disaccharide unitwithin the heparosan-based polysaccharide can be described by itsbackbone structure and by the number and position of sulfo groups thatare present. Further, the relative abundance of disaccharide unitshaving the same structure within the same polysaccharide, and/or withinthe same sample of polysaccharides, can be characterized to determinethe amount of sulfation at a particular position as a result of reactingwith any of the sulfotransferases described herein.

The terms, “fragment” or “segment,” refer to a polypeptide that has anamino- or carboxy-terminal deletion, but where the remaining amino acidsequence is identical to the corresponding positions in a referencesequence. Fragments can be at least 50 amino acids or longer, and up to70%, 80%, 90%, 95%, 98%, and 99% of a full-length aryl sulfate-dependentor natural sulfotransferase enzyme.

The terms, “functional site” or “functional domain,” generally refer toany site in a protein that confers a function on the protein.Representative examples include active sites (i.e., those sites incatalytic proteins where catalysis occurs) and ligand binding sites.Ligand binding sites include, but are not limited to, metal bindingsites, co-factor binding sites, antigen binding sites, substratechannels and tunnels, and substrate binding domains. In an enzyme, aligand binding site that is a substrate binding domain may also be anactive site. Functional sites may also be composites of multiplefunctional sites, wherein the absence of one or more sites comprisingthe composite results in a loss of function. As a non-limiting example,the active site of a particular sulfotransferase enzyme may includemultiple binding sites or clefts, including one site for the sulfo donorand one site for the sulfo acceptor.

The terms, “gene,” “gene sequence,” and “gene segment,” refer to afunctional unit of nucleic acid unit encoding for a functional protein,polypeptide, or peptide. As would be understood by those skilled in theart, this functional term includes both genomic sequences and cDNAsequences. The terms, “gene,” “gene sequence,” and “gene segment,”additionally refer to any DNA sequence that is substantially identicalto a polynucleotide sequence disclosed herein encoding for engineeredenzyme gene product, protein, or polysaccharide, and can comprise anycombination of associated control sequence. The terms also refer to RNA,or antisense sequences, complementary to such DNA sequences. As usedherein, the term “DNA segment” includes isolated DNA molecules that havebeen isolated free of recombinant vectors, including but not limited toplasmids, cosmids, phages, and viruses.

The term, “glycosaminoglycan,” refers to long, linear polysaccharidesconsisting of repeating disaccharide units. Examples ofglycosaminoglycans (GAGs) include chondroitin, dermatan, heparosan,hyaluronic acid, and keratan. GAGs are generally heterogeneous withrespect to mass, length, disaccharide unit structure andfunctionalization, degree of sulfation.

The term, “heparosan,” refers to a particular GAG having repeating[β(1,4)GlcA-α(1,4)GlcNAc]_(n) disaccharide units, in which GlcA isglucuronic acid and GlcNAc is N-acetyl glucosamine.

The term, “heparosan-based polysaccharide,” refers to polysaccharideshaving the same backbone structure as heparosan, in which thedisaccharide unit contains 1→4 glycosidically-linked hexuronic acid andglucosamine residues. The hexuronic acid residue can either be GlcA, asin heparosan, or iduronic acid (IdoA), and can optionally have a sulfogroup at the 2-O position. The glucosamine residue can either beN-acetylated, as in heparosan, N-sulfated, or N-unsubstituted, and canoptionally be sulfated at the N-, 3-O, or 6-O position. As used herein,the term “N-unsubstituted,” with respect to a glucosamine residue, isequivalent to an “N-deacetylated” glucosamine residue, and refers to anamine functional group that is capable of receiving a sulfo group eitherchemically, or enzymatically using an NST enzyme. According to thepresent invention, heparosan-based polysaccharides can be utilized asstarting materials, formed as intermediates, acting as sulfo groupacceptors and/or synthesized as products according to any of the methodsdescribed herein.

The term, “insertion,” refers to modifications to the polypeptide byaddition of one or more amino acids to the reference polypeptide.Insertions can be in the internal portions of the polypeptide, or to theC- or N-termini of the polypeptide. Insertions can include fusionproteins as is known in the art and described below. The insertions cancomprise a continuous segment of amino acids or multiple insertionsseparated by one or more of the amino acids in the referencepolypeptide.

The term, “isolated nucleic acid” as used herein with respect to nucleicacids derived from naturally-occurring sequences, means a ribonucleic ordeoxyribonucleic acid which comprises a naturally-occurring nucleotidesequence and which can be manipulated by standard recombinant DNAtechniques, but which is not covalently joined to the nucleotidesequences that are immediately contiguous on its 5′ and 3′ ends in thenaturally-occurring genome of the organism from which it is derived. Asused herein with respect to synthetic nucleic acids, the term “isolatednucleic acid” means a ribonucleic or deoxyribonucleic acid whichcomprises a nucleotide sequence which does not occur in nature and whichcan be manipulated by standard recombinant DNA techniques. An isolatednucleic acid can be manipulated by standard recombinant DNA techniqueswhen it may be used in, for example, amplification by polymerase chainreaction (PCR), in vitro translation, ligation to other nucleic acids(e.g., cloning or expression vectors), restriction from other nucleicacids (e.g., cloning or expression vectors), transformation of cells,hybridization screening assays, or the like.

The term, “low molecular weight heparin” refers to a natural orsynthesized heparin composition, generally having a weight averagemolecular weight less than 12,000 Da, and typically either prepared bydepolymerizing unfractionated heparin or API heparin, or by chemically,enzymatically, or chemoenzymatically synthesizing the polysaccharides denovo.

The terms, “naturally occurring” or “natural,” refer to forms of anenzyme found in nature. For example, a naturally occurring or naturalpolypeptide or polynucleotide sequence is a sequence present in anorganism that can be isolated from a source in nature and which has notbeen intentionally modified by human manipulation. A natural polypeptideor polynucleotide sequence can also refer to recombinant proteins ornucleic acids that can be synthesized, amplified, and/or expressed invitro, and which have the same sequence and biological activity as anenzyme produced in vivo. In contrast to naturally occurring or naturalsulfotransferase enzymes, the engineered aryl sulfate-dependentsulfotransferase enzymes utilized in accordance with methods of thepresent invention have different amino acid and nucleic acid sequences,biological activity with aryl sulfate compounds instead of PAPS as sulfogroup donors, and cannot be found in nature.

The term, “oligosaccharide,” refers to saccharide polymers containing asmall number, typically three to nine, sugar residues within eachmolecule.

The term, “percent identity,” refers to a quantitative measurement ofthe similarity between two or more nucleic acid or amino acid sequences.As a non-limiting example, the percent identity can be assessed betweentwo or more engineered enzymes of the present invention, two or morenaturally occurring enzymes, or between one or more engineered enzymesand one or more naturally occurring enzymes. Percent identity can beassessed relative to two or more full-length sequences, two or moretruncated sequences, or a combination of full-length sequences andtruncated sequences.

The term, “polysaccharide,” refers to polymeric carbohydrate structuresformed of repeating units, typically monosaccharide or disaccharideunits, joined together by glycosidic bonds, and which can range instructure from a linear chain to a highly-branched three-dimensionalstructure. Although the term “polysaccharide,” as used in the art, canrefer to saccharide polymers having more than ten sugar residues permolecule, “polysaccharide” is used within this application to describesaccharide polymers having more than one sugar residue, includingsaccharide polymers that have three to nine sugar residues that may bedefined in the art as an “oligosaccharide.” According to the presentinvention, the term “polysaccharide,” is also used to generally describeGAGs and GAG-based compounds, including chondroitin, dermatan,heparosan, hyaluronic acid, and keratan compounds.

The terms, “protein,” “gene product,” “polypeptide,” and “peptide” canbe used interchangeably to describe a biomolecule consisting of one ormore chains of amino acid residues. In addition, proteins comprisingmultiple polypeptide subunits (e.g., dimers, trimers or tetramers), aswell as other non-proteinaceous catalytic molecules will also beunderstood to be included within the meaning of “protein” as usedherein. Similarly, “protein fragments,” i.e., stretches of amino acidresidues that comprise fewer than all of the amino acid residues of aprotein, are also within the scope of the invention and may be referredto herein as “proteins.” Additionally, “protein domains” are alsoincluded within the term “protein.” A “protein domain” represents aportion of a protein comprised of its own semi-independent folded regionhaving its own characteristic spherical geometry with hydrophobic coreand polar exterior.

The term, “recombinant,” when used with reference to, for example, acell, nucleic acid, or polypeptide, refers to a material that has beenmodified in a manner that would not otherwise exist in nature.Non-limiting examples include, among others, recombinant cellsexpressing genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise expressed ata different level.

The term, “reference sequence,” refers to a disclosed or definedsequence used as a basis for sequence comparison. A reference sequencemay be a subset of a larger sequence, for example, a segment of afull-length gene or polypeptide sequence. Generally, a referencesequence refers to at least a portion of a full-length sequence,typically at least 20 amino acids, or the full-length sequence of thenucleic acid or polypeptide.

The term, “saccharide,” refers to a carbohydrate, also known as a sugar,which is a broad term for a chemical compound comprised of carbon,hydrogen, and oxygen, wherein the number of hydrogen atoms isessentially twice that of the number of oxygen atoms. Often, the numberof repeating units may vary in a saccharide. Thus, disaccharides,oligosaccharides, and polysaccharides are all examples of chainscomposed of saccharide units that are recognized by the engineeredsulfotransferase enzymes of the present invention as sulfo groupacceptors.

The term, “substantially equivalent,” with respect to polysaccharidesutilized as starting materials, formed as intermediates, acting as sulfogroup acceptors, and/or synthesized as products according to any of themethods described herein, refers to one or more properties of apolysaccharide sample that are identical to those found in apolysaccharide sample characterized in the prior art. Such propertiesmay include, but are not limited to, chemical structure, sulfationfrequency and location, disaccharide unit composition, molecular weightprofile, and/or anticoagulant activity. Even if the two polysaccharidesamples have additional properties that may be different, suchdifferences do not significantly affect their substantial equivalence.In a non-limiting example, anticoagulant N,2,3,6-HS products synthesizedaccording to methods of the present invention can be substantiallyequivalent to the United States Pharmacopeia (USP) reference standard(CAS No: 9041-08-1) with respect to chemical structure, molecular weightprofile, and/or anticoagulant activity, but can be produced at adifferent purity than the USP reference standard, which is isolated fromnatural sources and can contain non-trace amounts of other GAGs in thesame sample.

The term, “substantially pure,” with respect to protein preparations,refers to a preparation which contains at least 60% (by dry weight) theprotein of interest, exclusive of the weight of other intentionallyincluded compounds. Particularly the preparation is at least 75%, moreparticularly at least 90%, and most particularly at least 99%, by dryweight the protein of interest, exclusive of the weight of otherintentionally included compounds. Purity can be measured by anyappropriate method, e.g., column chromatography, gel electrophoresis, orhigh-performance liquid chromatography (HPLC) analysis. If a preparationintentionally includes two or more different proteins of the invention,a “substantially pure” preparation means a preparation in which thetotal dry weight of the proteins of the invention is at least 60% of thetotal dry weight, exclusive of the weight of other intentionallyincluded compounds. Particularly, for such preparations containing twoor more proteins of the invention, the total weight of the proteins ofthe invention can be at least 75%, more particularly at least 90%, andmost particularly at least 99%, of the total dry weight of thepreparation, exclusive of the weight of other intentionally includedcompounds.

The terms, “sulfo” or “sulfuryl” refer to a functional group,substituent, or moiety having the chemical formula SO₃H⁻ that can beremoved from an aryl sulfate compound and/or be transferred from a donorcompound to an acceptor compound. In some embodiments, the engineeredsulfotransferases of the present invention catalyze the transfer ofsulfo groups from aryl sulfate compounds to a polysaccharide,particularly heparosan and/or heparosan-based polysaccharides.

The term, “sulfotransferase,” refers to any enzyme in an in vivo or invitro process that is used to catalyze the transfer of a sulfo groupfrom a sulfo donor compound to a sulfo acceptor compound.“Sulfotransferase” can be used interchangeably to describe enzymes thatcatalyze sulfotransfer reactions in vivo or to describe engineeredenzymes of the present invention that catalyze sulfotransfer reactionsin vitro.

The term, “transformation,” refers to any method of introducingexogenous a nucleic acid into a cell including, but not limited to,transformation, transfection, electroporation, microinjection, directinjection of naked nucleic acid, particle-mediated delivery,viral-mediated transduction or any other means of delivering a nucleicacid into a host cell which results in transient or stable expression ofsaid nucleic acid or integration of said nucleic acid into the genome ofsaid host cell or descendant thereof.

The term, “unfractionated heparin,” refers to any synthesized orisolated heparin that has not been modified and/or partiallydepolymerized to form low molecular weight heparin. With respect tonaturally-obtained heparin, the term “unfractionated heparin” generallyrepresents the form of the heparin isolated from the animal, typicallyfrom porcine or bovine sources, prior to purification to meet USPreference standards. With respect to products synthesized by methods ofthe present invention, the term “unfractionated heparin” can refer tothe N,2,3,6-HS product having polysaccharides comprising thepentasaccharide sequence of Formula I, prior to purification to form APIheparin or low-molecular-weight heparin.

DETAILED DESCRIPTION OF THE INVENTION

In nature, heparosan is synthesized in the Golgi apparatus asco-polymers of glucuronic acid and N-acetylated glucosamine, beforebeing modified by one or more sulfotransferases to form heparan sulfate(HS) products. Such modifications include N-deacetylation andN-sulfation of glucosamine, C₅ epimerization of glucuronic acid to formiduronic acid residues, 2-O-sulfation of iduronic and/or glucuronicacid, as well as 6-O-sulfation and 3-O-sulfation of glucosamineresidues. The natural sulfotransferases that catalyze N-sulfation,2-O-sulfation, 6-O-sulfation and 3-O-sulfation of heparosan and HSpolysaccharides in vivo exclusively recognize and bind with PAPS, anearly ubiquitous sulfo group donor recognized by nearly allsulfotransferases, particularly in eukaryotes. An example of asulfotransfer reaction mechanism between the human glucosaminyl 3-Osulfotransferase (3OST) enzyme, PAPS, and heparan sulfate is illustratedin FIGS. 1A-1C. In particular, the glutamic acid residue at position 43abstracts the proton from the 3-O position of the N-sulfoglucosamineresidue within the polysaccharide, enabling the nucleophilic attack andremoval of the sulfo group from PAPS, whereas His-45 and Asp-48coordinate to stabilize the transition state of the enzyme before thesulfated polysaccharide product is released from the active site.

However, although PAPS is the exclusive sulfo donor in eukaryotes, ithas a short half-life and can readily decompose into adenosine3′,5′-diphosphate, which acts as a competitive inhibitor duringsulfotransfer reactions. Animals can efficiently utilize PAPS becausethey can metabolize adenosine 3′,5′-diphosphate to prevent competitiveinhibition and also replenish PAPS for each sulfotransfer reaction, asneeded. On the other hand, aryl sulfate compounds, which can be utilizedas sulfo donors in a limited number of bacterial systems (see Malojcic,G., et al., above), cannot react with any of the known nativesulfotransferase enzymes in eukaryotes, including those that areinvolved in synthesizing HS polysaccharides in vivo. Without beinglimited by a particular theory, it is believed that the binding pocketsfor PAPS within the active sites of eukaryotic sulfotransferases eitherdo not have a high enough affinity for aryl sulfate compounds tofacilitate binding, and/or that the aryl sulfate compounds aresterically hindered from entering the active site at all.

The present disclosure includes methods and kits for synthesizingsulfated polysaccharides, particularly HS polysaccharides, usingsulfotransferase enzymes that are engineered to recognize and bind witharyl sulfate compounds as sulfo group donors. Particularly, theengineered sulfotransferase enzymes are designed to transfer sulfogroups from aryl sulfate compounds to heparosan-based polysaccharides,containing alternating polymers of 1→4 glycosidically-linked hexuronicacid and glucosamine residues, to form HS polysaccharides. In vivo, HSpolysaccharides play critical roles in a variety of important biologicalprocesses, including assisting viral infection, regulating bloodcoagulation and embryonic development, suppressing tumor growth, andcontrolling the eating behavior of test subjects by interacting withspecific regulatory proteins. Depending on the role, HS polysaccharidescan contain one or more unique patterns or motifs recognized by specificprotein(s) involved in the particular biological process. The HSpolysaccharide produced by any of the methods or kits described hereincan have anticoagulant activity either analogous or identical tonaturally-sourced heparin.

It should be understood that while reference is made to exemplaryembodiments and specific language is used to describe them, nolimitation of the scope of the invention is intended. Furthermodifications of the methods described herein, as well as additionalapplications of the principles of those inventions as described, whichwould occur to one skilled in the relevant art and having possession ofthis disclosure, are to be considered within the scope of thisinvention. Furthermore, unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which embodiments ofthis particular invention pertain. The terminology used is for thepurpose of describing those embodiments only, and is not intended to belimiting unless specified as such. Headings are provided for convenienceonly and are not to be construed to limit the invention in any way.Additionally, throughout the specification and claims, a given chemicalformula or name shall encompass all optical isomers and stereoisomers,as well as racemic mixtures where such isomers and mixtures exist.

In Vitro Synthesis of Heparan Sulfate Polysaccharides

In an embodiment of the invention, the synthesis of HS polysaccharidescan be accomplished by treating a heparosan-based polysaccharide with anaryl sulfate compound and a sulfotransferase enzyme that has beenengineered to recognize, bind, and react with aryl sulfate compounds assulfo group donors. Each of the engineered sulfotransferase enzymes,including their sequences, structures, and biological activities, aredescribed in further detail below. Without being limited by a particulartheory, it is believed that sulfotransferase enzymes that recognizepolysaccharides as sulfo group acceptors, but also bind and react witharyl sulfate compounds as sulfo donors, have neither been observed innature nor described previously.

Those skilled in the art will appreciate that the engineeredsulfotransferase enzymes utilized in the methods of the presentinvention have several advantages over in vitro and in vivo reactionmechanisms that are unable to bind and react with aryl sulfate compoundsin order to catalyze sulfo transfer. Presently, obtaining large-scalequantities of sulfated polysaccharides, including heparin, requiresisolating and purifying them from animal sources, such as pigs andcattle (see Xu, Y., et al., (2011) Science 334 (6055): 498-501).However, a worldwide contamination crisis of heparin in 2007 and 2009shone a spotlight on the fragility of solely relying on obtaining themfrom animal sources. Consequently, in recent years, there has been apush to develop synthetic routes to synthesizing anticoagulant HSpolysaccharides in large enough quantities to compliment or replaceanimal-sourced products.

In order to synthesize sulfated polysaccharides in vitro, there havehistorically been two reaction schemes: total chemical synthesis andchemoenzymatic synthesis. While both types of reaction schemes have ledto purified products that in some instances are homogeneous, syntheticroutes as a whole have been inadequate to produce sulfatedpolysaccharides, particularly heparin, on an industrial scale. Indeed,the production of such polysaccharides using total chemical synthesishas historically required as many as 60 steps and resulted in very lowyields (see Balagurunathan, K., et al., (eds.) (2015)Glycosaminoglycans: Chemistry and Biology, Methods in Molecular Biology,vol. 1229, DOI 10.1007/978-1-4939-1714-3_2, © Springer Science+BusinessMedia New York).

Chemoenzymatic synthesis routes, on the other hand, generally utilizefar fewer steps and increase the scale of the generated anticoagulantproducts into multi-milligram amounts (See U.S. Pat. Nos. 8,771,995 and9,951,149, the disclosures of which are incorporated by reference in itsentirety). The improvements in the quantity of obtainable product can beattributed to the ability to combine recombinant naturalsulfotransferases and PAPS in a reaction vessel in order to catalyzesulfo group transfer. Yet, chemoenzymatic methods to this point areinadequate for forming heparin on a large scale, because of the naturalsulfotransferases' requirement to react with PAPS. PAPS is a highlyexpensive and unstable molecule that has been an obstacle to thelarge-scale production of enzymatically sulfated products, includingheparin, because the half-life of PAPS at pH 8.0 is only about 20 hours.

Furthermore, product inhibition by adenosine 3′,5′-diphosphate, which isa product of the sulfotransfer reaction, has also been a limiting factorto large-scale synthesis of heparin. The highly negative impact of theproduct inhibition by adenosine 3′,5′-diphosphate can be somewhatreduced by employing a PAPS regeneration system (see U.S. Pat. No.6,255,088, above, and Burkhart, et al. (2000) J. Org. Chem. 65:5565-5574) that converts adenosine 3′,5′-diphosphate into PAPS. Despitethe PAPS regeneration system, however, the absolute necessity to supplyPAPS to initiate the chemical reaction with native sulfotransferasesnonetheless creates an insurmountably high-cost barrier to synthesizesulfated products, including heparin, on an industrial, production-gradescale.

In contrast to prior chemoenzymatic syntheses of sulfatedpolysaccharides that require PAPS as a sulfo donor in order to driveactivity, the methods of the present invention obviate the need to usePAPS altogether, because each of the sulfotransferases have beenengineered to recognize, bind, and react with aryl sulfate compounds assulfo donors. As described above, some aryl sulfate compounds, such asPNS or MUS, are cheap, widely-available, and have previously been shownto react with some bacterial sulfotransferases as sulfo group donors(see Malojcic, G., et al., above). However, bacterial sulfotransferasesare unsuitable to synthesize heparin or any other sulfatedpolysaccharide, because bacterial sulfotransferases can only react withother aromatic compounds as substrates, and cannot bind or react withpolysaccharides. Consequently, and without being limited by a particulartheory, it is believed that the engineered sulfotransferases utilized inmethods of the present invention are the only known sulfotransferasesthat are capable of catalyzing sulfo group transfer from an aryl sulfatecompound to a polysaccharide, particularly heparosan-basedpolysaccharides. Generally, any of the methods described herein forsynthesizing sulfated products can be performed using one or moreengineered sulfotransferases, and such engineered sulfotransferases cancomprise any amino acid sequence so long as its biological activity isdependent on transferring a sulfo group from an aryl sulfate compound toheparosan-based polysaccharide. Non-limiting examples of engineeredenzymes, aryl sulfate compounds, and heparosan-based polysaccharides aredescribed in further detail, below.

In nature, heparan sulfate can be sulfated at the 2-O position of anyhexuronic acid residue and the N-, 3-O, 6-O position of any glucosamineresidue within the polysaccharide. Further, several of the hexuronicacid or glucosamine residues within the same polysaccharide chain can besulfated at any of the above positions, and can form a characteristicsulfation pattern that can be recognized by one or more enzymes orco-factors within the body. As a non-limiting example, heparin containspolysaccharides having a characteristic pentasaccharide sequence with aspecific sulfation pattern that is recognized by antithrombin.

In an embodiment of the invention, methods are provided forchemoenzymatically synthesizing N-, 2-O-, 3-O-, 6-O-sulfated-HS(N,2,3,6-HS) products, particularly heparin. One or more, and preferablyall, of the N-, 2-O-, 3-O-, and 6-O sulfation steps can be catalyzedusing sulfotransferase enzymes that are engineered to react with arylsulfate compounds in the absence of PAPS. Each of these enzymes aredescribed in further detail below. By controlling the molecular weightand N-acetyl glucosamine content of heparosan-based polysaccharidesutilized as starting materials, an N,2,3,6-HS product composition can beformed that has a comparable molecular weight, sulfation, andanticoagulant activity to the United States Pharmacopeia (USP) referencestandard (CAS No: 9041-08-1) for API heparin.

Heparin produced in vitro and in vivo contains heparan sulfatepolysaccharides having a consensus pentasaccharide motif, which can onlybe formed when sulfated in a specific order. Thus, in methods of thepresent invention in which a heparin product is synthesized, the orderof sulfation within the pentasaccharide motif is typically: (1)N-sulfation; (2) 2-O-sulfation; (3) 6-O-sulfation; and (4) 3-Osulfation. However, other portions of the polysaccharide can be sulfatedin any order, and other N,2,3,6-HS products can be synthesized bysulfating heparosan-based polysaccharides in any order. Each of thereaction steps utilized to synthesize any N,2,3,6-HS product, includingheparin, can optionally be performed in a single pot, or performed inone or more separate steps in which the products are isolated andpurified prior to performing the next sulfation step.

In general, and as described above, a vast majority of naturalsulfotransferases, including all sulfotransferases known to react withpolysaccharides, react with PAPS as a sulfo donor. Consequently, eachsulfotransferase enzyme is generally classified by the chemical reactionit catalyzes, particularly the sulfo group acceptor and thesubsequently-formed product. With respect to sulfotransferases thatreact with heparosan-based polysaccharides, the enzymes must furtherrecognize specific structural motifs and sulfation patterns within thepolysaccharide chain in order to bind and react. Each of the engineered,aryl sulfate-dependent sulfotransferases, and the sulfo acceptorpolysaccharides that they recognize, bind, and react with, are describedin further detail below.

Glucosaminyl N-sulfotransferases

In nature, N-sulfation is typically carried out byN-deacetylase/N-sulfotransferase (NDST) enzymes have dual activity, inwhich the same enzyme can catalyze the N-deacetylation of N-acetylglucosamine residues and the N-sulfation of unsubstituted glucosamineresidues within heparosan. In particular, N-sulfation is accomplished bythe enzymatic transfer of a sulfo group from PAPS to the glucosamineresidue. The dual N-deacetylase and N-sulfotransferase activity of NDSTis achieved via two separate structural domains—an N-deacetylase domainand an N-sulfotransferase domain. However, the activity of one of thedomains is not a pre-requisite for the activity of the other domain, andrecombinant single domain proteins comprising either N-deacetylase orN-sulfotransferase activity can be expressed and purified. Thus, in invitro syntheses of heparan sulfate products, a single-domain,recombinant N-sulfotransferase enzyme is often utilized to carry out theN-sulfation step. Similarly, and in an embodiment of the invention,engineered aryl sulfate-dependent NST enzymes can be expressed andpurified to comprise a single, N-sulfotransferase domain, in order tocatalyze the N-sulfation of N-deacetylated heparosan in the absence ofPAPS.

Naturally-occurring NDST enzymes, which react with PAPS as a sulfo groupdonor, are members of the EC 2.8.2.8 enzyme class. N-deacetylatedportions of heparosan that can react with natural NDST enzymes,recombinant N-sulfotransferase domains of natural NDST enzymes, and theengineered aryl-sulfate dependent NST enzymes described herein cancomprise one or more disaccharide units comprising the structure ofFormula II, below:

wherein n is an integer and R is selected from the group consisting of ahydrogen atom or a sulfo group. Although the portion of thepolysaccharide that reacts with the enzyme comprises the structure ofFormula II, other portions of the polysaccharide can be N- orO-substituted. Typically, N-deacetylated heparosan comprising thestructure of Formula II can comprise at least four disaccharide units,or eight sugar residues total. Sulfotransfer reactions in whichN-deacetylated heparosan is utilized as the sulfo group acceptor arediscussed in Sheng, J., et al., (2011) J. Biol. Chem. 286 (22):19768-76,as well as Gesteira, T. F., et al., (2013) PLoS One 8 (8):e70880, thedisclosures of which are incorporated by reference in their entireties.

Upon successfully binding PAPS and N-deacetylated heparosan, NDSTenzymes can catalyze transfer of the sulfo group to an unsubstitutedglucosamine, forming an N-sulfated heparosan product comprising thestructure of Formula III, below:

wherein n is an integer and R is selected from the group consisting of ahydrogen atom or a sulfo group. Similarly, when an engineered arylsulfate-dependent NST enzyme successfully binds with an aryl sulfatecompound and N-deacetylated heparosan, N-sulfation is catalyzed to forman N-sulfated heparosan product comprising the structure of Formula III.

In another embodiment, each of the repeating disaccharide units withinthe N-deacetylated heparosan that reacts with any of the natural NDSTenzymes or any of the engineered aryl sulfate-dependent NST enzymescomprises the structure of Formula II. In further embodiments, both ofthe R groups at the 6-O position of the glucosaminyl residues and the2-O position of the glucuronic acid residues are hydrogen atoms, in allof the disaccharide units. In other embodiments, in some locationswithin the polysaccharide, at least a portion of the glucosamineresidues are still N-acetylated, as shown in FIG. 2 , althoughglucosaminyl residues that are N-acetylated cannot directly participateas sulfo group acceptors. However, the presence of N-acetylated residueswithin the polysaccharide does not affect the sulfotransferases' bindingaffinity for non-acetylated residues within the same polysaccharide. Inanother embodiment, regardless of the structure of the heparosan-basedpolysaccharide adjacent to portion comprising the structure of FormulaII, the N-sulfated polysaccharide product generated by reacting with anengineered NST or natural NDST (or the recombinant N-sulfotransferasedomain of NDST) comprises the structure of Formula III.

In another embodiment, when there are multiple dimers comprising thestructure of Formula II within the polysaccharide, any unsubstitutedglucosamine residue can be N-sulfated. Similarly, the samepolysaccharide can be N-sulfated multiple times, including and up to allavailable unsubstituted glucosaminyl residues that are present withinthe chain.

In another embodiment, heparosan-based polysaccharides comprising thestructure of Formula II can be provided as a homogenous composition. Instill other embodiments, sulfo acceptor polysaccharides comprising thestructure of Formula II can be comprised within a composition comprisinga polydisperse mixture of polysaccharides having variable chain lengths,molecular weights, and monosaccharide composition and functionalization.

In another embodiment, heparosan-based polysaccharides comprising thestructure of Formula II and utilized in accordance with methods of thepresent invention can be obtained and/or modified from commercialsources. In other embodiments, heparosan can be isolated from bacterialor eukaryotic sources and subsequently chemically treated in order toproduce an N-deacetylated polysaccharide that comprises the structure ofFormula II. Such processes are discussed in detail in the descriptionand examples, below.

The N-sulfotransferase domains of natural NDST enzymes within EC 2.8.2.8typically comprise approximately 300 to 350 amino acid residues that canvary greatly in their sequence, yet ultimately have the exact samefunction, namely, to catalyze the N-sulfation of unsubstitutedglucosamine residues within N-deacetylated heparosan. Without beinglimited by a particular theory, it is believed that each of the naturalN-sulfotransferase domains can catalyze the same chemical reactionbecause there are multiple amino acid sequence motifs and secondarystructures that are either identical or highly conserved across allspecies.

Further, it is believed that several of the conserved amino acidsequence motifs within NDST are directly involved in binding of eitherPAPS and/or the polysaccharide, or participate in the chemical reactionitself. The identity of conserved amino acid sequence motifs between theNDST enzymes can be demonstrated by comparing the amino acid sequence ofthe N-sulfotransferase domain of the human NDST1 enzyme, which has asolved crystal structure (PDB code: 1NST) in which amino acid residueswithin the active site have been identified, with the amino acidsequences of the N-sulfotransferase domains of other natural NDSTs. Amultiple sequence alignment of the N-sulfotransferase domains of fifteenenzymes within EC 2.8.2.8, including several eukaryotic organisms andseveral isoforms of the human NDST, is shown in FIGS. 3A-3C, along withtheir percent identity relative to the human NDST1 (UniProtKB AccessionNo. P52848). As illustrated in FIGS. 3A-3C, sequences range from having98.4% sequence identity with the P52848 reference sequence (entrysp|Q02353|NDST1_RAT) for the rat N-sulfotransferase domain down to 55.6%sequence identity (entry sp|Q9V3L1|NDST_DROME) for the fruit flyN-sulfotransferase domain. Those skilled in the art would appreciatethat the multiple sequence alignment was limited to fifteen sequencesfor clarity, and that there are hundreds of amino acid sequencesencoding for the N-sulfotransferase domains of other natural NDSTenzymes that have been identified and that have highly conserved activesite and/or binding regions as well.

Within FIGS. 3A-3C, amino acids that are depicted in white with a blackbackground at a particular position, are 100% identical across allsequences. Amino acids that are highly conserved, meaning that the aminoacids are either identical or chemically or structurally similar, at aparticular position are enclosed with a black outline. Within highlyconserved regions, consensus amino acids that are present in a majorityof the sequences, are in bold. Amino acids at a particular position thatare not identical or highly conserved are typically variable. A periodwithin a sequence indicates a gap that has been inserted into thesequence in order to facilitate the sequence alignment with othersequence(s) that have additional residues between highly conserved oridentical region. Finally, above each block of sequences are a series ofarrows and coils that indicate secondary structure that is conservedacross all sequences, based on the identity of the amino acids withinthe alignment and using the structure of the natural humanN-sulfotransferase enzyme as a reference. The β symbol adjacent to anarrow refers to a β-sheet, whereas a coil adjacent to an α symbol or a ηsymbol refers to a helix secondary structure.

Within the fifteen aligned sequences in FIGS. 3A-3C, there are severalconserved amino acid motifs that include one or more amino acids thatcomprise the active site, based on the crystal structure of theN-sulfotransferase domain of human NDST1. These conserved amino acidsequence motifs, based on the numbering of the amino acid residueswithin FIGS. 3A-3C include residues 40-46 (Q-K-T-G-T-T-A); residues66-69 (T-F-E-E); residues 101-105 (F-E-K-S-A); residues 139-143(S-W-Y-Q-H); and residues 255-262 (C-L-G-K/R-S-K-G-R). In furtherembodiments, some isoforms of the natural sulfotransferase enzymeswithin EC 2.8.2.8 that comprise the conserved amino acid sequence motifQ-K-T-G-T-T-A further comprise the expanded conserved amino acidsequence motif, Q-K-T-G-T-T-A-L-Y-L, from residues 40-49.

Without being limited by a particular theory, it is believed that theseresidues either facilitate or participate in the chemical reaction, orenable binding of PAPS or the polysaccharide within the active site. Inparticular and as illustrated in FIGS. 4A-4C, the histidine residue atposition 143 (corresponding to position 716 in the amino acid sequenceof the full-length natural sulfotransferase enzyme that also includes anN-deacetylase domain) is in position to abstract one of the two protonswithin the amine functional group of the unsubstituted glucosaminylresidue within the polysaccharide, enabling the nitrogen atom toinitiate the nucleophilic attack of PAPS and remove the sulfuryl group.Additionally, lysine residues at position 41 and 260 are alsouniversally conserved, and are thought to coordinate with the sulfurylmoiety, driving binding of PAPS within the active site as well asstabilizing the transition state during the course of the reaction (seeGesteira, T. F., et al., above, as well as Sueyoshi, T., et al., (1998)FEBS Letters 433:211-214, the disclosure of which is incorporated byreference in its entirety).

However, as described above, the natural NDST enzymes are unable tocatalyze the transfer of the sulfate group from an aryl sulfate compoundto the polysaccharide, because without being limited by a particulartheory, it is believed that the binding pocket for PAPS either does nothave a high enough affinity for aryl sulfate compounds to facilitatebinding and/or that the aryl sulfate compounds are sterically hinderedfrom entering the active site. Consequently, and in another embodiment,the N-sulfotransferase domain of a natural NDST enzyme can be mutated inseveral locations within its amino acid sequence to enable binding ofthe aryl sulfate compound within the active site and/or to optimallyposition the aryl sulfate compound so transfer of the sulfate group tothe polysaccharide can occur.

Accordingly, and in another embodiment, engineered NST enzymes that canbe utilized in accordance with methods of the present invention cancomprise a single N-sulfotransferase domain that is mutated relative tothe N-sulfotransferase domain of any NDST enzyme, including enzymeshaving the amino acid sequences illustrated in FIGS. 3A-3C. In otherembodiments, engineered NST enzymes that can be utilized in accordancewith methods of the present invention can further comprise anN-deacetylase domain that has an identical or mutated amino acidsequence of the N-deacetylase domain of any natural NDST enzyme.

In another embodiment, mutations engineered into the amino acidsequences of the engineered enzymes facilitate a biological activity inwhich aryl sulfate compounds can both bind and react with the engineeredNST enzymes as sulfo group donors. In further embodiments, theengineered NST enzyme can bind and react with an aryl sulfate compoundas a sulfo group donor, while retaining the corresponding naturalsulfotransferases' biological activity with heparosan and/orN-deacetylated heparosan as a sulfo group acceptor. Without beinglimited by a particular theory, it is believed that because of themutations inserted into the amino acid sequences of the engineered NSTenzymes, their sulfotransferase activity may comprise the directtransfer of a sulfuryl group from an aryl sulfate compound to the sulfoacceptor polysaccharide, using a similar mechanism as described in FIGS.4A-4C above, except that the PAPS is substituted with the aryl sulfatecompound. Otherwise, it is believed that the mutations may cause thesulfotransferase activity to comprise a two-step process including thehydrolysis of an aryl sulfate compound and formation of a sulfohistidineintermediate, followed by the nucleophilic attack of the sulfohistidineintermediate by an N-unsubstituted glucosamine within N-deacetylatedheparosan to form the N-sulfated product. By either mechanism,engineered NST enzymes have been shown to achieve sulfo transfer from anaryl sulfate compound to a polysaccharide, as described in the examples,below.

In another embodiment, an engineered NST enzyme can comprise one or moremutated amino acid sequence motifs relative to the conserved amino acidsequence motifs described above that are found in the N-sulfotransferasedomains of natural NDST enzymes within EC 2.8.2.8, as described aboveand indicated in the multiple sequence alignment in FIG. 3 . In anotherembodiment, each mutated amino acid sequence motif that is present inthe amino acid sequence of the engineered NST enzyme comprises at leastone amino acid mutation relative to the corresponding conserved aminoacid sequence motif within the N-sulfotransferase domain of a naturalNDST. In another embodiment, an engineered NST enzyme comprises onemutated amino acid sequence motif. In another embodiment, an engineeredNST enzyme comprises two mutated amino acid sequence motifs. In anotherembodiment, an engineered NST enzyme comprises three mutated amino acidsequence motifs. In another embodiment, an engineered NST enzymecomprises four mutated amino acid sequence motifs. In anotherembodiment, an engineered NST enzyme comprises five mutated amino acidsequence motifs. In another embodiment, an engineered NST enzyme thatincludes at least one mutated amino acid sequence motif relative to anN-sulfotransferase domain of any of the natural NDST enzymes within EC2.8.2.8 can have an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQID NO: 40.

In another embodiment, upon viewing the crystal structure of theN-sulfotransferase domain of the human NDST1 (PDB code: 1NST) within a3D molecular visualization system (including, as a non-limiting example,the open-source software, PyMOL), the structure of related sequences,such as those of engineered NST enzymes that contain one or more mutatedamino acid sequence motifs relative to the human N-sulfotransferasedomain, can be modeled for comparison as illustrated in FIGS. 5-8 . Inone non-limiting example, FIG. 5 shows a magnified view of the activesite of the human N-sulfotransferase domain that is overlaid with anengineered NST enzyme, comprising the amino acid sequence of SEQ ID NO:10, in which the structure of the engineered enzyme is modelled uponmaking mutations relative to the human N-sulfotransferase domain aminoacid sequence. Adenosine 3′,5′-diphosphate, which is the product of asulfotransfer reaction in which PAPS is the sulfo donor, and which wasco-crystallized with the human N-sulfotransferase domain, is alsoillustrated within the active site. PNS is also modeled into theengineered enzyme active site, using the consensus solutions ofmolecular dynamics (MD) simulations that designed to calculate theoptimized position and orientation of a ligand within an enzyme activesite adjacent to the polysaccharide binding site (not shown), if suchsolutions are possible.

As illustrated in FIG. 5 , although there are several mutations withinSEQ ID NO: 10, relative to sequence of the human N-sulfotransferasedomain (UniProtKB Accession No. P52848) indicated in FIG. 3 , therespective protein backbones are in a nearly identical location to oneanother, enabling a one-to-one comparison of the active sites. Withinthe structure of the engineered enzyme comprising the sequence of SEQ IDNO: 10, the consensus solutions from MD simulations indicate that thesulfate moiety within PNS is favored to bind adjacent to a histidineresidue, His-45, that has been mutated relative to the natural aminoacid residue, threonine, which is also universally conserved within EC2.8.2.8. On the other hand, within the human N-sulfotransferase domain,the adenosine 3′,5′-diphosphate is located near to the conservedHis-143, described above. Although the sulfo group that would becomprised within the PAPS substrate is not shown, those skilled in theart would appreciate that if PAPS were present, the sulfate group wouldbe oriented in a position immediately adjacent to His-143 and partiallyoverlapping with the sulfate group within PNS. Without being limited bya particular theory, it is believed that the nearly overlapping locationof the sulfate groups accounts for the engineered enzyme's ability tofacilitate sulfo group transfer by using His-143 as a base to remove theproton from the glucosaminyl residue within the polysaccharide.

However, even though the sulfate groups can bind in a nearly identicallocation within the active site, aryl sulfate compounds cannot beutilized with EC 2.8.2.8 enzymes to facilitate sulfo group transfer to apolysaccharide. As described above, the amino acid residues within theactive site of the natural enzymes are evolved to have strong bindingaffinity for PAPS, and likely do not have enough affinity for arylsulfate compounds to drive binding and subsequently, reactivity.Consequently, other mutations must be present within the engineeredenzymes to drive binding of aryl sulfate compounds within the activesite. FIG. 6 illustrates other mutations that surround PNS within theengineered enzyme comprising the amino acid sequence of SEQ ID NO: 10,including Trp-106, His-69, and His-40. Trp-106 and His-69 are positionedto provide π-π stacking binding contacts with aromatic moiety withinPNS. Additionally, the ε2 nitrogen atoms within His-69 and His-40coordinate with the sulfuryl group directly. Lysine residues retainedfrom the natural enzyme sequence, Lys-41 (not shown, for clarity) andLys-103 are in position to coordinate with the sulfate group duringtransfer in order to stabilize the transition state. Of note, thenatural amino acid residue, Lys-260, which also coordinates with thesulfate group in PAPS, is mutated to a valine residue within theengineered enzyme sequence. Without being limited by a particulartheory, it is believed that His-45, which is necessary for the reactionwith PNS, would exhibit charge repulsion with a lysine residue atposition 260, and that the mutation to a valine residue retains somesteric bulk within the binding site while eliminating the chargerepulsion. Lys-103 is nonetheless positioned to coordinate with thesulfuryl group, particularly when the sulfuryl group is associated orbound to His-45, as shown in FIG. 6 .

In another non-limiting example, FIG. 7 shows a magnified view of theactive site of the human N-sulfotransferase domain (UniProtKB AccessionNo. P52848) that is overlaid with a different engineered NST enzyme,comprising the amino acid sequence of SEQ ID NO: 2. PNS is modeled intothe engineered enzyme active site, as described above. As with theengineered NST having the amino acid sequence SEQ ID NO: 10, the proteinbackbone of the enzyme having the amino acid sequence SEQ ID NO: 2 alsohas a nearly identical structure to the N-sulfotransferase domain of thehuman enzyme. However, the consensus solutions from MD simulationsindicate that the sulfate moiety within PNS is favored to bind adjacentto a different histidine mutation (His-49), which is mutated from anatural leucine residue that is conserved in the active site of theN-sulfotransferase domain of several of the natural NDST enzymes.Consequently, mutations within SEQ ID NO: 10 that formed bindingcontacts with PNS are not necessarily present in SEQ ID NO: 2. Asillustrated in FIG. 8 and similar to SEQ ID NO: 10, there are twomutations present within SEQ ID NO: 2 that appear to form π-π stackingbinding contacts surrounding the aromatic moiety of PNS, Trp-45 andHis-67. Other mutations that comprise side chains that coordinate withPNS include Ser-69 (coordinating with the nitro functional group of PNS)and His-260 (coordinating with the sulfate moiety). Similar to SEQ IDNO: 10, because the natural lysine residue at position 260 is mutated,the natural Lys-103 residue is utilized within SEQ ID NO: 2 tocoordinate with the sulfate moiety within PNS.

Those skilled in the art would appreciate that engineered NST enzymes ofany other amino acid sequence, including, but not limited to, thosedescribed by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12,SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, would likelyexhibit a similar structure to the human N-sulfotransferase domain andengineered NST enzymes having the amino acid sequence of SEQ ID NO: 2and SEQ ID NO: 10. Without being limited by a particular theory, it isalso believed that NCS would bind in a similar position as PNS withinthe active site of any of the engineered NST enzymes, since thestructures of the two aryl sulfate compounds are very similar, exceptthat the sulfate group is located ortho on the aromatic ring relative tothe nitro group, rather than para to the nitro group.

Further, engineered NST enzymes utilized in accordance with methods ofthe present invention can include mutated amino acid sequence motifsthat include the above-described mutations as well as other mutationsthat facilitate binding of substrates, the sulfotransfer reaction, orthe stability of the enzyme during protein expression. In anotherembodiment, an engineered NST enzyme can include the mutated amino acidsequence motif, X₁-K-T-G-A-W/F-A/L-L-X₂-H, mutated from the conservedamino acid sequence Q-K-T-G-T-T-A-L-Y-L within EC 2.8.2.8, wherein X₁ isselected from the group consisting of glutamine, serine, and alanine;and X₂ is selected from the group consisting of tyrosine, threonine, andhistidine. Engineered NST enzymes that include the mutated amino acidsequence motif X₁-K-T-G-A-W/F-A/L-L-X₂-H include, but are not limited toSEQ ID NO: 2 (described above), as well as SEQ ID NO: 4, SEQ ID NO: 12;SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 40. Infurther embodiments, engineered NST enzymes can further include themutated amino acid sequence motif, T-X₃-X₄-S, mutated from the conservedamino acid sequence T-F-E-E, wherein X₃ is a mutation relative to thenatural sulfotransferase enzymes within EC 2.8.2.8, selected from thegroup consisting of histidine and glycine; X₄ is a mutation relative tothe natural sulfotransferase enzymes within EC 2.8.2.8, selected fromthe group consisting of glycine, histidine, and serine; and wherein atleast one of X₃ and X₄ is a histidine residue. In some even furtherembodiments, X₁ is glutamine, X₂ is tyrosine, X₃ is histidine, X₄ isglycine, and the engineered NST enzyme further comprises the mutatedamino acid sequence motif, C-L-G-K/R-S-H-G-R. In other even furtherembodiments, X₁ is serine, X₂ is threonine, X₃ is glycine, X₄ ishistidine, and the engineered NST enzyme further comprises the mutatedamino acid sequence motif, C-H-G-K/R-R-W-G-R. In sill other even furtherembodiments, X₁ is alanine, X₂ is histidine, X₃ is histidine, X₄ isserine, and the engineered NST enzyme further comprises the mutatedamino acid sequence motif, C-A-H-K/R-G-L-G-R.

In another embodiment, engineered NST enzymes can include the mutatedamino acid sequence motif, H-X₅-T-G-X₆-H-A, mutated from the conservedamino acid sequence Q-K-T-G-T-T-A, wherein X₅ is selected from the groupconsisting of lysine and glycine; and X₆ is a mutation relative to thenatural sulfotransferase enzymes within EC 2.8.2.8, selected from thegroup consisting of glycine and valine. Engineered NST enzymes thatinclude the mutated amino acid sequence motif H-X₅-T-G-X₆-H-A include,but are not limited to SEQ ID NO: 10 (described above), as well as SEQID NO: 6, SEQ ID NO: 8; SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, andSEQ ID NO: 39. In further embodiments, X₅ is glycine and X₆ is glycine.In some even further embodiments, the engineered NST enzyme furthercomprises the mutated amino acid sequence motif, C-G-G-K/R-H-L-G-R. Inother even further embodiments, the engineered NST enzyme furthercomprises the mutated amino acid sequence motif, F-E-H-S-G.

In another embodiment, within any of the engineered NST enzymes thatinclude the mutated amino acid sequence motif, H-X₅-T-G-X₆-H-A, X₅ isselected from the group consisting of lysine and glycine; and X₆ is amutation relative to the natural sulfotransferase enzymes within EC2.8.2.8, selected from the group consisting of glycine and valine. Infurther embodiments, X₅ is selected to be lysine, X₆ is selected to bevaline, and the engineered NST enzyme further comprises the mutatedamino acid sequence motif, T-G-N-H.

Furthermore, the amino acid sequences (SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12) of six engineeredNST enzymes, which have been experimentally determined to be active witharyl sulfate compounds as sulfo group donors (see Example 2 below) canbe compared with the amino acid sequence of the N-sulfotransferasedomain of the human NDST1 (entry sp|P52848|NDST1_HUMAN) in a multiplesequence alignment to determine if there are relationships betweenmutations among each of the enzymes. A period within the amino acidsequence of an engineered enzyme indicates identity at a particularposition with the human N-sulfotransferase domain. As shown in FIG. 9 ,the sequence alignment demonstrates that while over 90% of the aminoacid residues within the six sulfotransferase sequences are identical,there are several positions in which multiple amino acids can be chosen.Without being limited by a particular theory, it is believed that theseenzymes have a similar relationship with each other as theN-sulfotransferase domains of the natural NDST enzymes that comprise EC2.8.2.8. As a result, and in another embodiment, engineered NST enzymescomprising an amino acid sequence in which multiple amino acids can bechosen at defined positions are disclosed as SEQ ID NO: 33 and SEQ IDNO: 34. Positions at which the identity of an amino acid can be chosenfrom a selection of possible residues are denoted in terms “Xaa,” “Xn,”or “position n,” where n refers to the residue position.

In another embodiment, within an engineered NST enzyme comprising theamino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34, the amino acidresidue at position 41 is lysine, the amino acid residue at position 44is alanine, the amino acid residue at position 45 is an aromatic aminoacid residue, preferably tyrosine or phenylalanine, and the amino acidresidue at position 49 is histidine. In another embodiment, when theengineered NST enzyme comprises the above residues from positions 41-49,the amino acid residue at position 67 is glycine or histidine, the aminoacid residue at position 68 is selected from the group consisting ofglycine, histidine, and serine, and the amino acid residue at position69 is serine.

In another embodiment, within an engineered NST enzyme comprising theamino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34, the amino acidresidue at position 40 is histidine and the amino acid residue atposition 45 is histidine. In further embodiments, the amino acid residueat position 41 is glycine and the amino acid residue at position 44 isglycine. In other further embodiments, the amino acid residue atposition 41 is lysine and the amino acid residue at position 44 isvaline. In even further embodiments, the amino acid residue at position67 is glycine and the amino acid residue at position 69 is histidine. Instill further embodiments, the amino acid residue at position 106 istryptophan. In even still further embodiments, the amino acid residue atposition 260 is valine.

In another embodiment, within an engineered NST enzyme comprising theamino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34, the amino acidsequence can optionally include one or more mutations at residuepositions not specified by an “Xn” or “Xaa,” so long as any suchmutations do not eliminate the NST and/or aryl sulfate-dependentactivity of the enzyme. In another embodiment, such mutations noteliminating aryl sulfate-dependent activity at positions not specifiedby an “Xn” or “Xaa” can include substitutions, deletions, and/oradditions.

Accordingly, in another embodiment, an engineered NST enzyme utilized inaccordance with any of the methods of the present invention can comprisean amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ IDNO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40. In anotherembodiment, any of the above enzymes react with an aryl sulfatecompound, instead of PAPS, as a sulfo group donor. In furtherembodiments, the aryl sulfate compound is selected from the groupconsisting of PNS, MUS, 7-hydroxycoumarin sulfate, phenyl sulfate,4-acetylphenyl sulfate, indoxyl sulfate, 1-naphthyl sulfate, 2-naphthylsulfate, and NCS. In some even further embodiments, the aryl sulfatecompound is PNS. In other even further embodiments, the aryl sulfatecompound is NCS.

Hexuronyl 2-O Sulfotransferases

In nature, HS hexuronyl 2-O sulfotransferase (2OST) enzymes recognize,bind, and react with N-sulfated heparosan-based polysaccharides as sulfogroup acceptors. As with the natural NDSTs described above, natural2OSTs transfer the sulfo group to the polysaccharide upon reacting withPAPS as a sulfo group donor. However, natural 2OSTs are members of theEC 2.8.2.- enzyme class. Generally, a majority of the glucosaminylresidues within the heparosan-based polysaccharide are N-sulfated, andthe sulfo group is transferred to the 2-O position of a hexuronic acidresidue, generally either glucuronic acid or iduronic acid. A firstnon-limiting example of an N-sulfated heparosan that can bind and reactwith a natural or engineered 2OST is illustrated by the structure ofFormula IV, below:

In another non-limiting example, an 2OST enzyme can recognize, bind, andreact with heparosan-based polysaccharides having the structure ofFormula V, below:

In both instances, the hexuronic acid residue (glucuronic acid inFormula IV, iduronic acid in Formula V) is flanked on either side byN-sulfated glucosamine residues that are otherwise unsubstituted at the3-O and 6-O positions. Natural 2OST enzymes, and their biologicalactivity with N-sulfated heparosan polysaccharides comprising thestructures of Formula IV or Formula V, have been described by Rong, J.,et al., (2001) Biochemistry 40 (18):5548-5555, the disclosure of whichis incorporated by reference in its entirety.

As described above, although the portion of the polysaccharide thatreacts with the enzyme comprises the structure of Formula IV or FormulaV, other portions can be N- or O-substituted. Similarly, theheparosan-based polysaccharides can comprise both the structure ofFormula IV and the structure of Formula V within the samepolysaccharide, and either or both of the hexuronyl residues within thestructure of Formula IV and Formula V polysaccharide can be sulfated bythe same enzyme molecule. Typically, N-sulfated HS polysaccharidescomprising the structure of Formula IV and/or Formula V can comprise atleast eight monosaccharide residues. In some embodiments, theheparosan-based polysaccharide is only N-sulfated or N-acetylated, andis not 3-O or 6-O sulfated prior to reacting with the 2OST. In anotherembodiment, engineered 2OSTs that can be utilized in accordance withmethods of the present invention have the same biological activity asnatural 2OSTs with heparosan-based polysaccharides, particularly thosecomprising the structure of Formula IV and Formula V, as sulfoacceptors.

The identity of the hexuronic acid residue in N-sulfated heparosancomprising the structure of Formula IV or Formula V can be controlled bythe presence of a hexuronyl C₅-epimerase, which reversibly inverts thestereochemistry of the C₅-carbon. However, once the hexuronyl residuewithin a polysaccharide comprising the structure of Formula IV orFormula V is 2-O sulfated, epimerization can no longer occur. Ineukaryotic systems, the N-sulfated heparosan products of NDST are almostexclusively formed as disaccharide units of N-sulfoglucosamine andglucuronic acid. Consequently, the glucuronic acid residue must beepimerized to an iduronic acid residue to from the structures of FormulaV prior to reacting with the 2OST enzyme. However, and without beinglimited by a particular theory, it is believed that natural 2OST enzymesgenerally have preference for binding and reacting with heparosan-basedpolysaccharides comprising the structure of Formula V, and that most N-,2-O sulfated HS (N,2-HS) polysaccharides produced in vivo generallycomprise 2-O sulfated iduronic acid.

Upon successfully binding PAPS and N-sulfated heparosan comprising thestructure of Formula IV, natural 2OST enzymes can catalyze transfer ofthe sulfo group to the 2-O position of the glucuronic acid residue,forming an N,2-HS product comprising the structure of Formula VI, below:

Similarly, engineered 2OST enzymes that successfully bind and react withan aryl sulfate compound and an N-sulfated heparosan comprising thestructure of Formula IV can also form an N,2-HS product comprising thestructure of Formula VI. Upon successfully binding PAPS and N-sulfatedheparosan comprising the structure of Formula V, natural 2OST enzymescan catalyze transfer of the sulfo group to the 2-O position of theiduronic acid residue, forming an N,2-HS product comprising thestructure of Formula VII, below:

Similarly, engineered 2OST enzymes that successfully bind and react withan aryl sulfate compound and an N-sulfated heparosan comprising thestructure of Formula V can also form an N,2-HS product comprising thestructure of Formula VII.

In another embodiment, in other locations within the N-sulfated sulfoacceptor polysaccharide, some of the glucosaminyl residues can beN-substituted with a sulfo group, an acetyl group, or a hydrogen,although hexuronyl residues within the polymer must reside between twoN-sulfoglucosamine residues, as described above, in order to receive asulfo group. A non-limiting example of one such polysaccharide isillustrated in FIG. 10 . In FIG. 10 , hexuronyl residues 10 withinpolysaccharide 40 are flanked by glucosaminyl residues 20, 21, and 22,that are either N-sulfated, N-acetylated, or unsubstituted,respectively. Upon reacting the polysaccharide with either a natural orengineered 2OST, only the hexuronyl residue 10 flanked by twoN-sulfoglucosaminyl residues 20 is sulfated, ultimately forming asulfated hexuronyl residue 110 within the product polysaccharide 41.

In another non-limiting example, sulfo acceptor polysaccharidescomprising the structures of Formula IV and Formula V are illustrated bypolysaccharide 50 in FIG. 11 , FIG. 12 , and FIG. 13 . Additionalmonosaccharide residues required for catalysis are omitted for clarity.In FIG. 11 , FIG. 12 , and FIG. 13 , a hexuronyl residue 10 and anepimerized hexuronyl residue 30 reside between the threeN-sulfoglucosaminyl residues 20 within polysaccharide 50. Althoughhexuronyl residues 10 and 30 are represented in a chair conformation,those skilled in the art can appreciate that such monosaccharideresidues within a longer oligo- or polysaccharide chain can adoptseveral different conformations, including chair, half-chair, boat,skew, and skew boat conformations, and that those additionalconformations are omitted for clarity.

Upon reacting polysaccharide 50 with any of the engineered arylsulfate-dependent 2OST enzymes that can be utilized with methods of thepresent invention, the enzyme can catalyze sulfo group transfer tohexuronyl residue 10 to form a sulfated hexuronyl residue 110 withinproduct polysaccharide 51 (FIG. 11 ), to epimerized hexuronyl residue 30to form a sulfated epimerized hexuronyl residue 130 within productpolysaccharide 52 (FIG. 12 ), or to both hexuronyl residue 10 andepimerized hexuronyl residue 30 to form a sulfated hexuronyl residue 110and a sulfated epimerized hexuronyl residue 130, respectively, withinproduct polysaccharide 53 (FIG. 13 ).

In another embodiment, polysaccharides comprising the structure ofFormula IV and/or Formula V can be provided as a homogenous composition.In still other embodiments, polysaccharides comprising the structure ofFormula IV and/or Formula V can be comprised within a compositioncomprising a polydisperse mixture of polysaccharides having variablechain lengths, molecular weights, relative abundance of Formula IVand/or Formula V, and overall monosaccharide composition andfunctionalization.

In some embodiments, polysaccharides comprising the structure of FormulaIV and/or Formula V and utilized in accordance with methods of thepresent invention can be obtained and/or modified from commercialsources. In other embodiments, polysaccharides comprising the structureof Formula IV and/or Formula V can be obtained by enzymatically orchemically N-sulfating polysaccharides isolated and modified frombacterial or eukaryotic sources. In still other embodiments,polysaccharides comprising the structure of Formula IV and/or Formula Vcan be obtained by isolating and purifying the sulfated polysaccharideproducts of any of the other engineered aryl sulfate-dependentsulfotransferases utilized in conjunction with methods of the presentinvention. Each of these processes are discussed in detail in thedescription and examples, below.

Natural 2OSTs within the EC 2.8.2.- enzyme class generally compriseapproximately 325-375 amino acid residues that in some cases varygreatly in their sequence, yet ultimately have the exact same function,namely, to catalyze the transfer of a sulfo group from PAPS to the 2-Oposition of hexuronyl residues within heparosan-based polysaccharides,particularly those comprising the structure of Formula IV and/or FormulaV. Without being limited by a particular theory, it is believed thateach of the natural 2OSTs can catalyze the same chemical reactionbecause there are multiple amino acid sequence motifs and secondarystructures that are either identical or highly conserved across allspecies.

Further, it is believed that several of the conserved amino acidsequence motifs are directly involved in binding of either PAPS and/orthe polysaccharide, or participate in the chemical reaction itself. Theidentity between the natural 2OST enzymes can be demonstrated bycomparing the amino acid sequence of enzymes with a known crystalstructure (e.g. chicken 2-O sulfotransferase, PDB codes: 3F5F and 4NDZ),in which amino acid residues within the active site have beenidentified, with the amino acid sequences of other 2OSTs within the EC2.8.2.- enzyme class. A multiple sequence alignment of twelve enzymes,including the chicken, human, and other 2OST enzymes, is shown in FIGS.14A-14D, along with percent identity relative to the chicken 2OSTreference sequence (UniProtKB Accession No. Q76KB1). As illustrated inFIGS. 14A-14D, sequences range from having 94.9% sequence identity withthe Q76KB1 reference sequence (entry tr|T1DMV2|T1DMV2_CROHD) for thetimber rattlesnake 2OST, down to 56.3% sequence identity (entrytr|0A131Z2T4| A0A131Z2T4_RHIAP) for the brown ear tick 2OST. The humanenzyme (entry sp|Q7LGA3|HS2ST_HUMAN) has 94.1% sequence identity withthe Q76KB1 reference sequence. Those skilled in the art would appreciatethat the multiple sequence alignment was limited to twelve sequences forclarity, and that there are hundreds of amino acid sequences encodingfor natural 2OST enzymes that have been identified and that have highlyconserved active site and/or binding regions as well.

Within FIGS. 14A-14D, amino acids that are depicted in white with ablack background at a particular position, are 100% identical across allsequences. Amino acids that are highly conserved, meaning that the aminoacids are either identical, or chemically or structurally similar, at aparticular position are enclosed with a black outline. Within highlyconserved regions, consensus amino acids that are present in a majorityof the sequences are in bold. Amino acids at a particular position thatare not identical or highly conserved are typically variable. A periodwithin a sequence indicates a gap that has been inserted into thesequence in order to facilitate the sequence alignment with othersequence(s) that have additional residues between highly conserved oridentical region. Finally, above each block of sequences are a series ofarrows and coils that indicate secondary structure that is conservedacross all sequences, based on the identity of the amino acids withinthe alignment and using the structure of the natural chicken 2OST enzymeas a reference. The β symbol adjacent to an arrow refers to a β-sheet,whereas a coil adjacent to an α symbol or a η symbol refers to a helixsecondary structure.

Within the twelve aligned sequences in FIGS. 14A-14D, there are severalconserved amino acid motifs that include one or more amino acids thatcomprise the active site, based on the crystal structures of the chicken2OST enzyme described above. Based on the numbering of the amino acidresidues within FIGS. 14A-14D, these motifs include residues 12-19(R-V-P-K-T-A/G-S-T), residues 40-44 (N-T-S/T-K-N), residues 71-74(Y-H-G-H), residues 108-115 (F-L-R-F/H-G-D-D/N-F/Y), residues 121-125(R-R-K/R-Q-G), and residues 217-222 (S-H-L-R-K/R-T). Without beinglimited by a particular theory, it is believed that these residueseither facilitate or participate in the chemical reaction, or enablebinding of PAPS or the polysaccharide within the active site. Inparticular and as illustrated in FIGS. 15A-15C, the histidine residue atposition 74 abstracts the proton from the 2-O position of the iduronicacid residue within the polysaccharide, enabling nucleophilic attack andremoval of the sulfo group from PAPS, whereas the lysine residue atposition 15 coordinates with the phosphate moiety of PAPS to stabilizethe transition state of the enzyme before the N,2-HS product is releasedfrom the active site.

However, as described above, the natural 2OST enzymes are unable tocatalyze the transfer of the sulfate group from an aryl sulfate compoundto the polysaccharide. As with the NDSTs, it is believed that thebinding pocket for PAPS within the active site of the naturalsulfotransferase either does not have a high enough affinity for arylsulfate compounds to facilitate binding and/or that the aryl sulfatecompounds are sterically hindered from entering the active site.Consequently, and in another embodiment, a natural 2OST enzyme can bemutated in several locations within its amino acid sequence to enablebinding of the aryl sulfate compound within the active site and/or tooptimally position the aryl sulfate compound so transfer of the sulfategroup to the polysaccharide can occur.

Accordingly, and in another embodiment, engineered 2OST enzymes that canbe utilized with methods of the present invention can be mutants ofnatural 2OST enzymes within EC 2.8.2.-, including enzymes having theamino acid sequences illustrated in FIGS. 14A-14D. In anotherembodiment, the aryl sulfate-dependent, 2OSTs have been engineered torecognize, bind, and react with aryl sulfate compounds as sulfo groupdonors, while retaining the natural enzymes' ability to recognize, bind,and react with N-sulfated, heparosan-based polysaccharides, particularlythose comprising the structure of Formula IV and/or Formula V, as sulfogroup acceptors. Without being limited by a particular theory, it isbelieved that because of the mutations inserted into the amino acidsequences of the engineered 2OST enzymes, their sulfotransferaseactivity may comprise the direct transfer of a sulfuryl group from anaryl sulfate compound to the sulfo acceptor polysaccharide, using asimilar mechanism as described in FIGS. 15A-15C above, except that thePAPS is substituted with the aryl sulfate compound. Otherwise, it isbelieved that the mutations may cause the sulfotransferase activity tocomprise a two-step process including the hydrolysis of an aryl sulfatecompound and formation of a sulfohistidine intermediate, followed by thenucleophilic attack of the sulfohistidine intermediate by the oxygenatom at the 2-O position of a hexuronic acid residue, to form the N,2-HSproduct. By either mechanism, engineered 2OST enzymes achieve sulfotransfer from an aryl sulfate compound to a polysaccharide, as describedin the examples, below.

In another embodiment, an engineered 2OST enzyme can comprise one ormore mutated amino acid sequence motifs relative to the conserved aminoacid sequence motifs described above that are found in the natural 2OSTenzymes within EC 2.8.2.-, as described above and indicated in themultiple sequence alignment in FIGS. 14A-14D. In another embodiment,each mutated amino acid sequence motif that is present in the amino acidsequence of the engineered enzyme comprises at least one amino acidmutation relative to the corresponding conserved amino acid sequencemotif within the natural 2OST enzymes. In another embodiment, anengineered 2OST enzyme can comprise one mutated amino acid sequencemotif. In another embodiment, an engineered 2OST enzyme can comprise twomutated amino acid sequence motifs. In another embodiment, an engineered2OST enzyme can comprise three mutated amino acid sequence motifs. Inanother embodiment, an engineered 2OST enzyme can comprise four mutatedamino acid sequence motifs. In another embodiment, an engineered 2OSTenzyme can comprise five mutated amino acid sequence motifs. In anotherembodiment, an engineered 2OST enzyme can comprise six mutated aminoacid sequence motifs. In another embodiment, an engineered 2OST enzymethat includes at least one mutated amino acid sequence motif relative toany of the natural 2OST enzymes within EC 2.8.2.- can have an amino acidsequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 41, and SEQ ID NO: 42.

In another embodiment, upon viewing the crystal structure of the chicken2OST (PDB code: 3F5F) within a 3D molecular visualization system(including, as a non-limiting example, the open-source software, PyMOL),the structure of related sequences, such as those of engineered 2OSTenzymes that contain one or more mutated amino acid sequence motifsrelative to the chicken sulfotransferase structure, can be modeled forcomparison as illustrated in FIG. 16 . FIG. 16 shows a magnified view ofthe active site of the chicken 2OST enzyme overlaid with two engineered2OST enzymes, comprising the amino acid sequences of SEQ ID NO: 14 andSEQ ID NO: 16, in which the structure of the engineered enzyme iscalculated upon making mutations relative to the chicken 2OST amino acidsequence. Adenosine 3′,5′-diphosphate, which is the product of asulfotransfer reaction in which PAPS is the sulfo donor, and which wasco-crystallized with the chicken 2OST, is also illustrated within theactive site. The sulfate group that would be present in the naturalsubstrate, PAPS, is modeled onto the 5′-phosphate functional group toillustrate its approximate position within the active site prior toinitiating the reaction. NCS is also modeled into the active site of theengineered enzymes, using the consensus solutions of molecular dynamics(MD) simulations that designed to calculate the optimized position andorientation of a ligand within an enzyme active site adjacent to thepolysaccharide binding site (not shown), if such solutions are possible.Hydrogen atoms are not shown.

As illustrated in FIG. 16 , although there are several mutations made toSEQ ID NO: 14 and SEQ ID NO: 16, relative to the chicken 2OST, therespective protein backbones are in a nearly identical location to oneanother, enabling a one-to-one comparison of the active sites. Whencomparing the two active sites, the PAPS is located in the backgroundand adjacent to a lysine residue (position 15 of the Q76KB1 sequence inFIGS. 14A-14D), whereas the convergent solutions from the above MDsimulations indicate that NCS binding within the engineered enzymes isfavored on the opposite side of the active site. However, binding of NCSwould be sterically hindered in the natural enzyme in part by the lysineresidue as well as the phenylalanine residue located on the nearbyα-helix (position 108 of the Q76KB1 sequence in FIGS. 14A-14D). Withoutbeing limited by a particular theory, it is believed that binding of NCSin the active site of the engineered enzyme comprising the amino acidsequence of SEQ ID NO: 14 is facilitated by the mutation of the lysineresidue to a histidine residue, which creates additional space withinthe active site and provides a π-π stacking partner for the aromaticring within NCS. Also without being limited by a particular theory, itis believed that binding of NCS in the active site of the engineeredenzyme comprising the amino acid sequence of SEQ ID NO: 16 isfacilitated by the mutation of the lysine to an arginine residue inconcert with the adjacent mutation of the proline residue (position 14of the Q76KB1 sequence in FIGS. 14A-14D) to a histidine residue. Theincreased number of conformational degrees of freedom of the arginineside chain facilitate entry of the NCS while still being in a positionto provide a polar contact to stabilize the transition state during thetransfer reaction, whereas the adjacent histidine provides other bindingcontacts for NCS.

Another mutation of note includes the mutation from an arginine residue(position 220 of the Q76KB1 sequence in FIGS. 14A-14D) to a histidineresidue, a mutation that is found at position 221 in both SEQ ID NO: 14and SEQ ID NO: 16. Without being limited by a particular theory, themutated histidine residue is in a favorable position to facilitateremoval of the sulfate group from NCS. Other illustrated mutations fromthe chicken 2OST enzyme, particularly mutations present in SEQ ID NO: 16(His-20, Ser-114, Lys-116, Met-122) may similarly drive binding of NCSwithin the active site, either by providing a direct binding contactwith the sulfate moiety within NCS (His-20), coordinating with othermutated residues (Ser-114 coordinating with His-221), or by increasingthe hydrophobic environment near NCS (Met-122).

Those skilled in the art would appreciate that engineered 2OST enzymesof any other amino acid sequence, including, but not limited to, thosedisclosed by SEQ ID NO: 41 and SEQ ID NO: 42, would likely exhibit asimilar structure to the chicken 2OST, as well as engineered 2OSTshaving the amino acid sequence of SEQ ID NO: 14 and SEQ ID NO: 16.Without being limited by a particular theory, it is believed that PNSwould bind in a similar position as NCS within the active site of any ofthe engineered 2OST enzymes, since the structures of the two arylsulfate compounds are very similar, except that the sulfate group islocated ortho on the aromatic ring relative to the nitro group in NCS,rather than para to the nitro group in PNS.

Accordingly, in another embodiment, an engineered 2OST enzyme utilizedin accordance with any of the methods of the present invention cancomprise an amino acid sequence selected from the group consisting ofSEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 41, or SEQ ID NO: 42. Inanother embodiment, any of the above 2OST enzymes react with an arylsulfate compound, instead of PAPS, as a sulfo group donor. In furtherembodiments, the aryl sulfate compound is selected from the groupconsisting of PNS, MUS, 7-hydroxycoumarin sulfate, phenyl sulfate,4-acetylphenyl sulfate, indoxyl sulfate, 1-naphthyl sulfate, 2-naphthylsulfate, and NCS. In some even further embodiments, the aryl sulfatecompound is PNS. In other even further embodiments, the aryl sulfatecompound is NCS.

In another embodiment, within reaction mixtures that comprise anynatural or engineered 2OST enzyme, particularly an engineered 2OSTenzyme comprising the amino acid sequence of SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 41, or SEQ ID NO: 42, the reaction mixture can furthercomprise an hexuronyl C₅-epimerase to catalyze formation of an N,2-HSproduct. In some embodiments, the N,2-HS product can comprise thestructure of Formula VI. In other embodiments, the N,2-HS product cancomprise the structure of Formula VII. In another embodiment, anyisolated or recombinant hexuronyl C₅-epimerase can be used. In anotherembodiment, the hexuronyl C₅-epimerase can comprise the amino acidsequence of SEQ ID NO: 29. In another embodiment, the hexuronylC₅-epimerase can comprise residues 34-617 of SEQ ID NO: 29.

Glucosaminyl 6-O Sulfotransferases

In nature, 6OSTs recognize, bind, and react with heparosan-basedpolysaccharides as sulfo group acceptors. Generally, a majority of theglucosaminyl residues are N-sulfated, but the enzymes can still transfersulfo groups to the 6-O position of glucosaminyl residues that areN-acetylated. Additionally, either adjacent hexuronic acid residue canbe either glucuronic acid or iduronic acid, and can optionally be 2-Osulfated. Generally, the hexuronic acid at the non-reducing end of theglucosamine residue receiving the 6-O sulfo group is 2-O sulfatediduronic acid, and in many instances, the glucosamine residue itself isalso N-sulfated. Similar to the NSTs and 2OSTs, naturally-occurring 6OSTenzymes transfer the sulfo group to the polysaccharide upon reactingwith PAPS as a sulfo group donor. As with natural 2OSTs, natural 6OSTenzymes are also members of the EC 2.8.2.-enzyme class. In anon-limiting example, either natural or engineered 6OST enzymes canrecognize, bind, and react with heparosan-based polysaccharidescomprising the structure of Formula VIII, below:

wherein the glucosamine residue receiving the 6-O sulfo group isN-sulfated and is adjacent to a 2-O sulfated iduronic acid residue atits non-reducing end, and X comprises any of the hexuronyl residuesdepicted in Formula VIII, above. 6OST enzymes within EC 2.8.2.- havingbiological activity with polysaccharides comprising the structure ofFormula VIII have been described by Xu, Y., et al., (2017) ACS Chem.Biol. 12 (1):73-82 and Holmborn, K., et al., (2004) J. Biol. Chem. 279,(41):42355-42358, the disclosures of which are incorporated by referencein their entireties.

As described above, although the portion of the heparosan-basedpolysaccharide that reacts with the 6OST enzyme can comprise thestructure of Formula VIII, other portions of the polysaccharide can beN- or O-substituted, and can comprise other structural motifs that canalso react with the enzyme. Similar to the other enzymes above, 6OSTenzymes can transfer a sulfo group to multiple positions within the samepolysaccharide molecule, and multiple positions within the samepolysaccharide molecule can be 6-O sulfated by the same enzyme molecule.Typically, heparosan-based polysaccharides that can react with 6OSTenzymes, including those comprising the structure of Formula VIII, cancomprise at least three monosaccharide residues.

Upon successfully binding PAPS and a heparosan-based polysaccharidecomprising the structure of Formula VIII, natural 6OST enzymes cancatalyze transfer of the sulfo group to the 6-O position of theglucosamine residue, forming an N,2,6-HS product comprising thestructure of Formula IX, below:

wherein X comprises any of the hexuronyl residues depicted in FormulaIX, above. Similarly, an engineered 6OST enzyme that binds and reactswith an aryl sulfate compound and a heparosan-based polysaccharidecomprising the structure of Formula VIII can form an N,2,6-HS productcomprising the structure of Formula IX.

A non-limiting example of one such polysaccharide sulfo acceptor thatcan react with an 6OST enzyme is illustrated in FIG. 17 . FIG. 17 showsa heparosan-based polysaccharide 240 that includes three N-substitutedglucosamine residues 210 that can be N-substituted with either an acetylgroup 211 or a sulfate group 212. Within the polysaccharide 240,N-substituted glucosamine residues 210 that are capable of acting as asulfo acceptor are flanked by two hexuronyl residues. Hexuronyl residuescan include any residue represented by the functional group “X” inFormula VIII, particularly glucuronyl residue 220 and iduronyl residue230. Either the glucuronyl residue 220 or iduronyl residue 230 canfurther be substituted by a sulfate group 231 at the 2-O position. Uponreacting the polysaccharide 240 with an 6OST enzyme and a sulfo groupdonor, the 6-O position 213 of any of the glucosamine residues 210 canbe sulfated, ultimately forming 6-O sulfated glucosamine residues 310within the product polysaccharide 241. In another embodiment, the 6OSTenzyme can be an engineered aryl sulfate-dependent enzyme, and the sulfogroup donor is an aryl sulfate compound.

In another embodiment, engineered 6OSTs that can be utilized inaccordance with methods of the present invention can have the samebiological activity with heparosan-based sulfo acceptor polysaccharidesas natural 6OSTs, particularly heparosan-based polysaccharidescomprising the structure of Formula VIII. In another embodiment, whenthere are multiple portions of the polysaccharide comprising thestructure of Formula VIII within the sulfo acceptor polysaccharide, anyglucosamine residue can be sulfated by the engineered 6OST enzyme.Similarly, the same polysaccharide can be sulfated multiple times by theengineered 6OST, including and up to all of the glucosamine residuesthat are present within the polysaccharide.

In another embodiment, sulfo acceptor polysaccharides that can reactwith an engineered or natural 6OST, including but not limited to thosecomprising the structure of Formula VIII, can be provided as ahomogenous composition. In still other embodiments, sulfo acceptorpolysaccharides that can react with an engineered or natural 6OST can becomprised within a composition comprising a polydisperse mixture ofpolysaccharides having variable chain lengths, molecular weights,relative abundance of Formula VIII, and overall monosaccharidecomposition and functionalization.

In another embodiment, N,2-HS polysaccharides, including but not limitedto those comprising the structure of Formula VIII, and utilized inaccordance with methods of the present invention with either anengineered or natural 6OST enzyme can be obtained and/or modified fromcommercial sources. In another embodiment, either an engineered ornatural 6OST can be utilized in accordance with methods of the presentinvention can react with N-sulfated heparosan products produced by anNST enzyme in one or more previous steps. In another embodiment, eitheran engineered or natural 6OST that can be utilized in accordance withmethods of the present invention can react with N,2-HS products producedby an NST and/or a 2OST in one or more previous steps. In anotherembodiment, one or more of the sulfation steps to produce the N,2-HSproduct was catalyzed by an engineered, aryl sulfate-dependentsulfotransferase. Each of these processes are discussed in detail in thedescription and examples, below.

Natural 6OST enzymes within the EC 2.8.2.- enzyme class generallycomprise between 300 and 700 amino acid residues that can in some casesvary greatly in their sequence, yet ultimately have the exact samefunction, namely, to catalyze the transfer of a sulfuryl group from PAPSto the 6-O position of glucosamine residues within heparosan-basedpolysaccharides, particularly those comprising the structure of FormulaVIII. Without being limited by a particular theory, it is believed thateach of the natural 6OSTs can catalyze the same chemical reactionbecause there are multiple amino acid sequence motifs and secondarystructures that are either identical or highly conserved across allspecies.

Further, it is believed that several of the conserved amino acidsequence motifs are directly involved in binding of either PAPS and/orthe polysaccharide, or participate in the chemical reaction itself. Theidentity between the natural 6OST enzymes can be demonstrated bycomparing the amino acid sequence of an enzyme with a known crystalstructure (zebrafish 6OST isoform 3-B, PDB codes 5T03, 5T05 and 5T0A),in which amino acid residues within the active site have beenidentified, with the amino acid sequences of other natural 6OSTs. Amultiple sequence alignment of fifteen enzymes is shown in FIGS.18A-18C, along with the percent identity of each sequence relative tothe mouse 6OST (isoform 1) reference sequence (UniProtKB Accession No.Q9QYK5). As illustrated in FIGS. 18A-18C, sequences range from having97.3% identity with the Q9QYK5 reference sequence (entryO60243|H6ST1_HUMAN) down to 53.7% identity (entryA0A3P8W3M9|A0A3P8W3M9_CYSNE). For comparison, the zebrafish 6OST isoform3-B enzyme (entry A0MGZ7|H6S3B_DANRE) has 60.4% sequence identity withthe Q9QYK5 reference sequence. Those skilled in the art would appreciatethat the multiple sequence alignment was limited to fifteen sequencesfor clarity, and that there are hundreds of amino acid sequencesencoding for natural 6OST enzymes that have been identified and thathave highly conserved active site and/or binding regions as well.

Within FIGS. 18A-18C, amino acids that are depicted in white with ablack background at a particular position, are 100% identical across allsequences. Amino acids that are highly conserved, meaning that the aminoacids are either identical or chemically or structurally similar, at aparticular position are enclosed with a black outline. Within highlyconserved regions, consensus amino acids that are present in a majorityof the sequences, are in bold. Amino acids at a particular position thatare not identical or highly conserved are typically variable. A periodwithin a sequence indicates a gap that has been inserted into thesequence in order to facilitate the sequence alignment with othersequence(s) that have additional residues between highly conserved oridentical region. Finally, above each block of sequences are a series ofarrows and coils that indicate secondary structure that is conservedacross all sequences, based on the identity of the amino acids withinthe alignment and using the structure of the natural mouse 6OST enzymesenzyme as a reference. The β symbol adjacent to an arrow refers to aβ-sheet, whereas a coil adjacent to an α symbol refers to a helixsecondary structure. Each of the fifteen aligned sequences inillustrated FIGS. 18A-18C have been truncated relative to their naturalfull-length sequences to coincide with the engineered enzymes of thepresent invention, particularly SEQ ID NO: 18, SEQ ID NO: 20, and SEQ IDNO: 22. In particular, the residues illustrated in FIGS. 18A-18C arealigned with residues 67-377 of the Q9QYK5 reference sequence for themouse 6OST.

Within the fifteen aligned sequences in FIGS. 18A-18C, there are severalconserved amino acid sequence motifs that include one or more aminoacids that comprise the active site, based on the crystal structure ofthe zebrafish 6OST enzyme (entry A0MGZ7|H6S3B_DANRE) described above.Based on the numbering of the amino acid residues within FIGS. 18A-18C,these conserved amino acid sequence motifs include amino acid residues29 through 34 (Q-K-T-G-G-T); 81 through 86 (C-G-L-H-A-D); 127 through139 (S-E-W-R/K-H-V-Q-R-G-A-T-W-K); 178 through 184 (N-L-A-N-N-R-Q); and227 through 231 (L-T-E-F/Y-Q). In particular, and as illustrated in thereaction mechanism in FIGS. 19A-19C, the histidine residue within theC-G-L-H-A-D conserved amino acid sequence motif is in position toabstract the hydrogen atom from the 6′ hydroxyl group of anN-sulfoglucosamine residue, enabling the negatively-charged oxygen atomto then initiate the nucleophilic attack of PAPS and remove the sulfategroup. Additionally, the universally conserved lysine residue within theQ-K-T-G-G-T conserved amino acid sequence motif coordinates with the5′-phosphate in PAPS, while the universally conserved histidine andtryptophan residues at positions 131 and 138 coordinate with theN-sulfoglucosamine residue (see Xu, Y., et al., above).

However, as described above, natural 6OST enzymes are unable to catalyzethe transfer of the sulfate group from an aryl sulfate compound to apolysaccharide. Without being limited by a particular theory, and aswith the NSTs and 2OSTs described above, it is believed that the bindingpocket for PAPS within the active site of the natural 6OST either doesnot have a high enough affinity for aryl sulfate compounds to facilitatebinding and/or that the aryl sulfate compounds are sterically hinderedfrom entering the active site. Consequently, and in another embodiment,a natural 6OST enzyme can be mutated in several locations within itsamino acid sequence to enable binding of the aryl sulfate compoundwithin the active site and/or to optimally position the aryl sulfatecompound so transfer of the sulfate group to the polysaccharide canoccur.

Accordingly, and in another embodiment, engineered 6OST enzymes that canbe utilized with methods of the present invention can be mutants ofnatural 6OST enzymes within EC 2.8.2.-, including enzymes having theamino acid sequences illustrated in FIGS. 18A-18C. In anotherembodiment, the engineered 6OST enzymes have been engineered torecognize, bind, and react with aryl sulfate compounds as sulfo groupdonors, while retaining the natural enzymes' ability to recognize, bind,and react with any of the HS polysaccharides described above, includingbut not limited to those comprising the structure of Formula VIII, assulfo group acceptors. Without being limited by a particular theory, itis believed that because of the mutations inserted into the amino acidsequences of the engineered 6OST enzymes, their sulfotransferaseactivity may comprise the direct transfer of a sulfuryl group from anaryl sulfate compound to the sulfo acceptor polysaccharide, using asimilar mechanism as described in FIGS. 19A-19C, above, except that thePAPS is substituted with the aryl sulfate compound. Otherwise, it isbelieved that the mutations may cause the sulfotransferase activity tocomprise a two-step process including the hydrolysis of an aryl sulfatecompound and formation of a sulfohistidine intermediate, followed by thenucleophilic attack of the sulfohistidine intermediate by the oxygenatom at the 6-O position of a glucosamine residue, to form a 6-Osulfated HS product. In another embodiment, the 6-O sulfated HS productof either sulfotransfer mechanism is an N,2,6-HS product.

In another embodiment, an engineered 6OST enzyme can comprise one ormore mutated amino acid sequence motifs relative to the conserved aminoacid sequence motifs found in natural 6OST enzymes within EC 2.8.2.-, asdescribed above and indicated in the multiple sequence alignment inFIGS. 18A-18C. In another embodiment, each mutated amino acid sequencemotif that is present in the amino acid sequence of the engineeredenzyme comprises at least one amino acid mutation relative to thecorresponding conserved amino acid sequence motif within the natural6OST enzymes. In another embodiment, an engineered 6OST enzyme cancomprise one mutated amino acid sequence motif. In another embodiment,an engineered 6OST enzyme can comprise two mutated amino acid sequencemotifs. In another embodiment, an engineered 6OST enzyme can comprisethree mutated amino acid sequence motifs. In another embodiment, anengineered 6OST enzyme can comprise four mutated amino acid sequencemotifs. In another embodiment, an engineered 6OST enzyme can comprisefive mutated amino acid sequence motifs. In another embodiment, anengineered 6OST enzyme that includes at least one mutated amino acidsequence motif relative to any of the natural 6OST enzymes within EC2.8.2.- can have an amino acid sequence selected from the groupconsisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, andSEQ ID NO: 61.

In another embodiment, upon viewing any of the crystal structures of thezebrafish 6OST (UniProtKB Accession No. A0MGZ7) within a 3D molecularvisualization system (including, as a non-limiting example, theopen-source software, PyMOL), the structure of related sequences, suchas those of engineered 6OST enzymes that contain one or more mutatedamino acid sequence motifs relative to any of the zebrafish 6OSTstructures, can be modeled for comparison as illustrated in FIG. 20 .FIG. 20 shows a magnified view of the active site of the zebrafish 6OSTenzyme (PDB code: 5T03) with one of the engineered enzymes of thepresent invention, comprising the amino acid sequence of SEQ ID NO: 22,in which the structure of the engineered 6OST enzyme is calculated uponmaking mutations relative to the zebrafish 6OST amino acid sequence.Adenosine 3′,5′-diphosphate, which is the product of a sulfotransferreaction in which PAPS is the sulfo donor, and which was co-crystallizedwith the zebrafish 6OST, is also illustrated within the active site. PNSis also modeled into the active site of the engineered enzymes, usingthe consensus solutions of molecular dynamics (MD) simulations thatdesigned to calculate the optimized position and orientation of a ligandwithin an enzyme active site adjacent to the polysaccharide binding site(not shown), if such solutions are possible. Hydrogen atoms are notshown for clarity.

As illustrated in FIG. 20 , although there are several mutations madeSEQ ID NO: 22, relative to the zebrafish 6OST enzyme, the respectiveprotein backbones are in a nearly identical location to one another,enabling a one-to-one comparison of the active sites. However, whencomparing the two active sites, the adenosine 3′,5′-diphosphate productis located on the opposite side of the central α-helix as the PNSmolecule, as determined by the convergent solutions from the above MDsimulations. Without being limited by a particular theory, it isbelieved that the convergent MD simulation solutions place PNS on theopposite side of the α-helix because there is not enough of an affinitytoward PNS in the same or similar position as PAPS within the zebrafishenzyme. As described by Xu, Y., et al., above, the conserved histidineat position 158 of the full-length amino acid sequence is the catalytichistidine that abstracts the proton from the 6′ hydroxyl group ofN-sulfoglucosamine, which is then subsequently able to react with PAPSto initiate sulfo group transfer. Yet, despite the apparent differencesin the binding pocket for PAPS and PNS, engineered 6OST enzymescomprising the amino acid sequences of SEQ ID NO: 18, SEQ ID NO: 20, andSEQ ID NO: 22 all achieved sulfo transfer from an aryl sulfate compoundto the glucosaminyl 6-O position within a heparosan-basedpolysaccharide, as described in the examples below.

As a result, and without being limited by a particular theory, one ormore mutations present within the active site of engineered 6OST enzymesmay assist binding of the sulfate moiety of the aryl sulfate compound ina position in which it can be transferred to the sulfo acceptor HSpolysaccharide. As illustrated in FIG. 20 , the engineered enzyme hasthe amino acid sequence SEQ ID NO: 22, and the aryl sulfate compound isPNS. However, a sulfo acceptor HS polysaccharide is not illustrated. Ina non-limiting example, the histidine residue engineered into position31 of SEQ ID NO: 22 may be in position to facilitate removal of thesulfate group from PNS using a ping-pong mechanism, as described inMalojcic, et al, above. Additionally, the histidine residue engineeredinto position 133 of SEQ ID NO: 22 may further coordinate with thesulfate moiety along with the conserved histidine at position 132 of SEQID NO: 22 (corresponding to positions 131-132 in each of the sequencesin FIGS. 18A-18C). Mutation to G-A-N at positions 137-139 of SEQ ID NO:22 (corresponding to the conserved A-T-W motif at positions 136-138 ofthe sequences in FIGS. 18A-18C) removes steric bulk that may preventbinding of PNS in a position where the sulfate can be abstracted by theengineered histidine at position 31 of SEQ ID NO: 22. The mutations toG-A-N within the loop containing A-T-W also appears to cause the loop tomove away from PNS, which may further assist PNS to reach its bindingpocket. Finally, a serine residue engineered into position 84 of SEQ IDNO: 22, immediately adjacent to a native histidine corresponding toHis-158 in the full-length zebrafish 6OST, described above, may createan additional hydrogen-binding contact to assist the engineered enzymein retaining the zebrafish enzyme's natural activity with the sulfoacceptor polysaccharide.

Those skilled in the art would appreciate that engineered 6OST enzymesof any other amino acid sequence, including, but not limited to, thosedisclosed by SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 43, SEQ ID NO: 44,SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO:49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61,would exhibit similar structural motifs, particularly within the activesite. Without being limited by a particular theory, it is believed thatNCS would bind in a similar position as PNS within the active site ofany of the engineered enzymes, since the structures of the two arylsulfate compounds are very similar, except that the sulfate group islocated ortho on the aromatic ring relative to the nitro group, ratherthan para to the nitro group.

In another embodiment, engineered 6OST enzymes that can be utilized inaccordance with methods of the present invention can comprise one ormore mutated amino acid sequence motifs, which can be determined in-partby comparing conserved amino acid sequence motifs indicated in themultiple sequence alignment of FIGS. 18A-18C with the known structure(s)of natural enzymes and/or modeled engineered enzymes, including but notlimited to, as a non-limiting example, enzymes illustrated in FIG. 20 .In another embodiment, mutated amino acid sequence motifs that can becomprised within an engineered 6OST enzyme can be selected from thegroup consisting of (a) G-H-T-G-G-T; (b) C-G-X₁-X₂-A-D, wherein X₁ isselected from the group consisting of threonine and serine, and X₂ isselected from the group consisting of asparagine, arginine, andhistidine; (c) X₃-X₄-W-R-H-X₅-Q-R-G-G-X₆-N-K, wherein X₃ is selectedfrom the group consisting of serine and glycine, X₄ is selected from thegroup consisting of glycine and histidine, X₅ is selected from the groupconsisting of histidine and threonine, and X₆ is selected from the groupconsisting of alanine and threonine; and (d) N-L-X₇-N-N-R-Q, wherein X₇is selected from the group consisting of alanine and glycine; includingany combination thereof. Each of the mutated amino acid sequence motifscorresponds with a conserved amino acid motif indicated in FIGS. 18A-18Cabove: sequence motif (a) corresponds to the conserved amino acidsequence motif, Q-K-T-G-G-T; mutated amino acid sequence motif (b)corresponds to the conserved amino acid sequence motif, C-G-L-H-A-D;mutated amino acid sequence motif (c) corresponds to the conserved aminoacid sequence motif, S-E-W-(R/K)-H-V-Q-R-G-A-T-W-K; and mutated aminoacid sequence motif (d) corresponds to the conserved amino acid sequencemotif, N-L-A-N-N-R-Q. In another embodiment, engineered 6OST enzymescomprising at least one mutated amino acid sequence motif describedabove can be selected from the group consisting of: SEQ ID NO: 18, SEQID NO: 20, SEQ ID NO: 22, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45,SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61.

In another embodiment and in one non-limiting example, engineered 6OSTenzymes can comprise the mutated amino acid sequence motifs (b) and (c)within the same amino acid sequence. Engineered enzymes comprising themutated amino acid sequence motifs (b) and (c) include, but are notlimited to, enzymes comprising the amino acid sequences of SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 43, SEQ ID NO: 44, SEQ IDNO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, andSEQ ID NO: 50. In another embodiment, each of the engineered 6OSTenzymes comprising the mutated amino acid sequence motifs (b) and (c)have a similar active site as SEQ ID NO: 22, as illustrated in FIG. 20 .Without being limited to another theory, it is believed that several ofthe mutations comprised within mutated amino acid sequence motifs (b)and (c) have one or more functions during sulfotransferase activity,including not limited to: increasing the affinity of aryl sulfatecompounds to the active site by reducing the size of the binding pocket,increasing the hydrophobicity of the pocket, removing or creating polaror hydrogen bonding contacts, and/or creating π-π interactions with thearomatic moieties of the aryl sulfate compounds; stabilizing thetransition state of the enzyme during the chemical reaction; and/orparticipating in the chemical reaction itself.

In another embodiment, within engineered 6OST enzymes that comprise themutated amino acid sequence motifs (b) and (c), X₄ is glycine and X₅ ishistidine. In other embodiments, X₄ is histidine and X₅ is threonine.

In another embodiment, within engineered 6OST enzymes comprising themutated amino acid sequence motifs (b) and (c), X₃ is serine, X₆ isalanine, and X₇ is glycine. In other embodiments, X₃ is glycine, X₆ isthreonine, and X₇ is alanine.

Furthermore, the amino acid sequences (SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22) of three engineered 6OST enzymes, which have beenexperimentally determined to be active with aryl sulfate compounds assulfo group donors (see Example 4 below) can be compared with the aminoacid sequence of the mouse 6OST enzyme (entry Q9QYK5|H6ST1_MOUSE) in amultiple sequence alignment to determine if there are relationshipsbetween mutations among each of the enzymes. A period within the aminoacid sequence of an engineered enzyme indicates identity at a particularposition with the mouse 6OST enzyme. As shown in FIG. 21 , the sequencealignment demonstrates that while over 90% of the amino acid residueswithin the three sulfotransferase sequences are identical, there areseveral positions in which multiple amino acids can be chosen. Withoutbeing limited by a particular theory, these enzymes have a similarrelationship with each other as the 6OST enzymes that comprise EC2.8.2.-. As a result, and in another embodiment, engineered 6OST enzymescomprising an amino acid sequence in which multiple amino acids can bechosen at defined positions are disclosed as SEQ ID NO: 43 and SEQ IDNO: 44. Positions at which the identity of an amino acid can be chosenfrom a selection of possible residues are denoted in terms “Xaa,” “Xn,”or “position n,” where n refers to the residue position.

In another embodiment, within SEQ ID NO: 43, residues having thedesignation, “Xaa,” illustrate known instances in which there is a lackof identity at a particular position within the amino acid sequences ofSEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. In another embodiment,the amino acid sequence, SEQ ID NO: 44, also illustrates known instancesin which there is a lack of identity at a particular position within theamino acid sequences of SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22,but SEQ ID NO: 44 further comprises N-terminal residues 1-66, andC-terminal residues 378-411, of several full-length 6OST enzymes withinEC 2.8.2.-, including, as non-limiting examples, the mouse, human, andpig 6OST enzymes. In contrast, amino acid residues in SEQ ID NO: 18, SEQID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 43 correspond with residues67-377 of several full-length 6OST enzymes within EC 2.8.2.-, including,as non-limiting examples, the mouse, human, and pig 6OST enzymes. Tofacilitate protein expression, an N-terminal methionine residue wasadded to each SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, and SEQ IDNO: 43 amino acid sequence, relative to residues 67-377 of the mouse,human, and pig 6OST enzymes.

In another embodiment, any selection can be made for an Xaa residue,defined by the amino acid sequence SEQ ID NO: 43 or SEQ ID NO: 44, solong as the resulting enzyme maintains its 6OST activity upon reactingwith an aryl sulfate compound as a sulfo group donor.

In another embodiment, within an engineered 6OST enzyme comprising theamino acid sequence of SEQ ID NO: 43, the amino acid residue at position129 is glycine and the amino acid residue at position 133 is histidine.In another embodiment, within an engineered 6OST enzyme comprising theamino acid sequence of SEQ ID NO: 43, the amino acid residue at position129 is histidine and the amino acid residue at position 133 isthreonine. In another embodiment, within an engineered 6OST enzymecomprising the amino acid sequence of SEQ ID NO: 44, the amino acidresidue at position 194 is glycine and the amino acid residue atposition 198 is histidine. In another embodiment, within an engineered6OST enzyme comprising the amino acid sequence of SEQ ID NO: 44, theamino acid residue at position 194 is histidine and the amino acidresidue at position 198 is threonine.

In another embodiment, within an engineered 6OST enzyme comprising theamino acid sequence of SEQ ID NO: 43, the amino acid residue at position128 is serine, the amino acid residue at position 138 is alanine, andthe amino acid residue at position 181 is glycine. In anotherembodiment, within an engineered 6OST enzyme comprising the amino acidsequence of SEQ ID NO: 43, the amino acid residue at position 128 isglycine, the amino acid residue at position 138 is threonine, and theamino acid residue at position 181 is alanine. In another embodiment,within an engineered 6OST enzyme comprising the amino acid sequence ofSEQ ID NO: 44, the amino acid residue at position 193 is serine, theamino acid residue at position 203 is alanine, and the amino acidresidue at position 246 is glycine. In another embodiment, within anengineered 6OST enzyme comprising the amino acid sequence of SEQ ID NO:44, the amino acid residue at position 193 is glycine, the amino acidresidue at position 203 is threonine, and the amino acid residue atposition 246 is alanine.

In another embodiment, within an engineered 6OST enzyme comprising theamino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 44, the amino acidsequence can optionally include one or more mutations at residuepositions not specified by an “Xn” or “Xaa,” so long as any suchmutations do not eliminate the 6OST and/or aryl sulfate-dependentactivity of the enzyme. In another embodiment, such mutations noteliminating aryl sulfate-dependent activity at positions not specifiedby an “Xn” or “Xaa” can include substitutions, deletions, and/oradditions.

Accordingly, in another embodiment, an engineered 6OST enzyme utilizedin accordance with any of the methods of the present invention cancomprise an amino acid sequence selected from the group consisting ofSEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61.In another embodiment, any of the above engineered 6OST enzymes reactwith an aryl sulfate compound, instead of PAPS, as a sulfo group donor.In further embodiments, the aryl sulfate compound is selected from thegroup consisting of PNS, MUS, 7-hydroxycoumarin sulfate, phenyl sulfate,4-acetylphenyl sulfate, indoxyl sulfate, 1-naphthyl sulfate, 2-naphthylsulfate, and NCS. In some even further embodiments, the aryl sulfatecompound is PNS. In other even further embodiments, the aryl sulfatecompound is NCS.

Glucosaminyl 3-O Sulfotransferases

In nature, 3OSTs generally recognize, bind, and react with N,2-HSpolysaccharides and N,2,6-HS polysaccharides as sulfo group acceptors.Generally, the glucosamine residue that receives the sulfo group at the3-O position is N-sulfated, and is optionally also 6-O sulfated.Additionally, either adjacent hexuronic acid residue can be eitherglucuronic acid or iduronic acid, and can optionally be 2-O sulfated. Insome embodiments, the hexuronic acid residue on the non-reducing end ofthe glucosamine residue is unsulfated glucuronic acid, while thehexuronic acid residue on the reducing end of the glucosamine residue is2-O sulfated iduronic acid. Similar to each of the naturalsulfotransferases described above, naturally-occurring 3OSTs transferthe sulfo group to the polysaccharide upon reacting with PAPS as a sulfogroup donor. Natural 3OST enzymes that utilize PAPS as the sulfo groupdonor are members of the EC 2.8.2.23 enzyme class. In a non-limitingexample, both natural 3OST enzymes and engineered aryl sulfate-dependent3OST enzymes can recognize, bind, and react with N,2,6-HSpolysaccharides comprising the structure of Formula X, below:

wherein the central glucosamine residue is N-sulfated and is adjacent toan unsubstituted glucuronic acid residue at its non-reducing end and a2-O sulfated iduronic acid residue at its reducing end, X can optionallybe a sulfate group or an acetyl group, and Y can optionally be a sulfategroup or a hydroxyl group.

As described above, although the portion of the polysaccharide thatreacts with the enzyme comprises the structure of Formula X, otherportions of the polysaccharide can be N- or O-substituted, and cancomprise other structural motifs that can also react with the enzyme.Similar to the other enzymes above, 3OST enzymes can transfer a sulfogroup to multiple positions within the same polysaccharide molecule, andmultiple positions within the same polysaccharide molecule can be 3-Osulfated by the same enzyme molecule. Typically, HS polysaccharides thatcan react with 3OSTs as sulfo group acceptors typically comprise atleast five monosaccharide residues, as shown in Formula X. In anotherembodiment, polysaccharides comprising the structure of Formula X andcan react with 3OSTs as sulfo group acceptors can comprise at least 32monosaccharide residues.

Upon successfully binding PAPS and an N,2,6-HS polysaccharide comprisingthe structure of Formula X, natural 3OST enzymes can catalyze transferof the sulfo group to the 3-O position of the central glucosamineresidue, forming an N,2,3,6-HS product comprising the structure ofFormula I, below:

wherein X is either a sulfo group or an acetate group and Y is either asulfo group or a hydroxyl group. Similarly, engineered 3OST enzymes thatreact with an aryl sulfate compound and an N,2,6-HS polysaccharidecomprising the structure of Formula X can also form an N,2,3,6-HSproduct comprising the structure of Formula I. In further embodiments,the functional group X in the N,2,3,6-HS product is a sulfate group. Inother further embodiments, the functional group Y in the N,2,3,6-HSproduct is a sulfate group. In another embodiment, in some locationswithin the polymer, at least a portion of the glucosamine residues areN-acetylated. Natural 3OST enzymes within EC 2.8.2.23, which havebiological activity with N,2,6-HS polysaccharides comprising thestructure of Formula X as sulfo group acceptors and form N,2,3,6-HSproducts comprising the structure of Formula I, have been described byXu, D., et al., (2008) Nat. Chem. Biol. 4(3): 200-202 and Edavettal, S.C., et al., (2004) J. Biol. Chem. 24(11): 25789-25797, the disclosuresof which are incorporated by reference in their entireties.

A non-limiting example of one such N,2,6-HS sulfo group acceptor for3OST enzymes is illustrated in FIG. 22 . FIG. 22 shows a polysaccharide440 that includes three glucosamine residues 410 comprising an N-sulfogroup 411 at each N-position and an O-sulfo group 412 at each 6-Oposition. Within the polysaccharide 440, glucosamine residues 410 thatare capable of acting as a sulfo acceptor must be flanked by twohexuronic acid residues. Hexuronic acid residues can include any residuerepresented by the functional group “X” in Formula X, and are shown inFIG. 22 as glucuronic acid residue 420 and iduronic acid residue 430.Either hexuronic acid residue can further be substituted by a sulfogroup 431 at the 2-O position. Upon reacting the polysaccharide 440 withan 3OST enzyme and a sulfo group donor, the 3-O position 413 of any ofthe glucosaminyl residues 410 can be sulfated. As shown in FIG. 22 , thecentral glucosamine residue 410 receives a sulfo group, ultimatelyforming a 3-O sulfated glucosaminyl residue 510 within the sulfatedproduct polysaccharide 441. Also as shown, sulfated productpolysaccharide 441 comprises the structure of Formula I.

In another embodiment, engineered 3OSTs that can be utilized inaccordance with methods of the present invention can have the samebiological activity with heparosan-based sulfo acceptor polysaccharidesas natural 3OSTs, particularly heparosan-based polysaccharidescomprising the structure of Formula X. In another embodiment, when thereare multiple portions of the polysaccharide comprising the structure ofFormula X within the sulfo acceptor polysaccharide, any N-sulfatedglucosamine residue can be 3-O sulfated by the engineered 3OST enzyme.Similarly, the same polysaccharide can be sulfated multiple times by theengineered 3OST, including and up to all of the N-sulfated glucosamineresidues that are present within the polysaccharide. In anotherembodiment, a heparin mixture, either isolated from an animal source orsynthesized according to any of the methods described herein, can alsobe utilized as a sulfo group acceptor and further 3-O sulfated uponreacting with an engineered 3OST enzyme and an aryl sulfate compound, toform an “over-sulfated” heparin mixture.

In another embodiment, sulfo acceptor polysaccharides that can reactwith an engineered or natural 3OST, including but not limited to thosecomprising the structure of Formula X, can be provided as a homogenouscomposition. In still other embodiments, sulfo acceptor polysaccharidesthat can react with an engineered or natural 3OST can be comprisedwithin a composition comprising a polydisperse mixture ofpolysaccharides having variable chain lengths, molecular weights,relative abundance of Formula X, and overall monosaccharide compositionand functionalization.

In another embodiment, N,2-HS and N,2,6-HS polysaccharides, includingbut not limited to those comprising the structure of Formula X, andutilized in accordance with methods of the present invention with eitheran engineered or natural 6OST enzyme, can be obtained and/or modifiedfrom commercial sources. In another embodiment, either an engineered ornatural 6OST can be utilized in accordance with methods of the presentinvention can react with N,2-HS products produced by an NST and/or a2OST in one or more previous steps. In another embodiment, either anengineered or natural 6OST can be utilized in accordance with methods ofthe present invention can react with N,2,6-HS products produced by anNST, a 2OST, and/or a 6OST in one or more previous steps. In anotherembodiment, one or more of the sulfation steps to produce the N,2-HS orN,2,6-HS product was catalyzed by an engineered, aryl sulfate-dependentsulfotransferase. In another embodiment, all of the sulfation steps toproduce the N,2-HS or N,2,6-HS product was catalyzed by an engineered,aryl sulfate-dependent sulfotransferase. Each of these processes arediscussed in detail in the description and examples, below.

Natural 3OST enzymes within the EC 2.8.2.23 enzyme class generallycomprise approximately 300 to 325 amino acid residues that can in somecases vary greatly in their sequence, yet ultimately have the exact samefunction, namely, to catalyze the transfer of a sulfuryl group from PAPSto the 3-O position of N-sulfoglucosamine residues within N,2-HS orN,2,6-HS polysaccharides, particularly those comprising the structure ofFormula X. Without being limited by a particular theory, it is believedthat each of the natural 3OSTs can catalyze the same chemical reactionbecause there are multiple amino acid sequence motifs and secondarystructures that are either identical or highly conserved across allspecies.

Further, it is believed that several of the conserved amino acidsequence motifs are directly involved in binding of either PAPS and/orthe polysaccharide, or participate in the chemical reaction itself. Theidentity between the natural 3OST enzymes can be demonstrated bycomparing the amino acid sequence of a particular enzyme with 3OSTenzymes that have known crystal structures in which amino acid residueswithin the active site have been identified, including the mouse (PDBcode: 3UAN) and human (PDB code: 1ZRH) 3OST1 enzymes, which have nearlyidentical active sites and overall structures even though they have onlyan 83% sequence identity with one another. A multiple sequence alignmentof fifteen enzymes within EC 2.8.2.23, including the mouse and humanenzymes, is shown in FIGS. 23A-23C, along with the percent identity ofeach sequence relative to a human 3OST reference sequence (UniProtKBAccession No. O14792). As illustrated in FIGS. 23A-23C, sequences rangefrom having 98% identity with the O14792 reference sequence (entrytr|H9ZG39|H9ZG39_MACMU) for the rhesus monkey 3OST, down to 53% identity(entry sp|Q8IZT8|HS3S5_HUMAN) for human 3OST5. Those skilled in the artwould appreciate that the multiple sequence alignment was limited tofifteen sequences for clarity, and that there are hundreds of amino acidsequences encoding for natural 3OST enzymes that have been identifiedand that have highly conserved active site and/or binding regions aswell.

Within FIGS. 23A-23C, amino acids that are depicted in white with ablack background at a particular position, are 100% identical across allsequences. Amino acids that are highly conserved, meaning that the aminoacids are either identical or chemically or structurally similar, at aparticular position are enclosed with a black outline. Within highlyconserved regions, consensus amino acids that are present in a majorityof the sequences, are in bold. Amino acids at a particular position thatare not identical or highly conserved are typically variable. A periodwithin a sequence indicates a gap that has been inserted into thesequence in order to facilitate the sequence alignment with othersequence(s) that have additional residues between highly conserved oridentical region. Finally, above each block of sequences are a series ofarrows and coils that indicate secondary structure that is conservedacross all sequences, based on the identity of the amino acids withinthe alignment and using the structure of the natural humansulfotransferase enzyme as a reference. The β symbol adjacent to anarrow refers to a β-sheet, whereas a coil adjacent to an α symbol or ηsymbol refers to a helix secondary structure.

Within the fifteen aligned sequences in FIGS. 23A-23C, there are severalconserved amino acid sequence motifs that include one or more aminoacids that comprise the active site, based on the crystal structures ofthe mouse (entry sp|O35310|HS3S1_MOUSE) and human 3OST1 (entrysp|O14792|HS3S1_HUMAN) enzymes described above. Based on the numberingof the amino acid residues within FIGS. 23A-23C, these motifs includeresidues 16-27 (including G-V-R-K-G-G from residues 18-23), residues43-48 (E-V/I-H-F-F-D), residues 78-81 (P-A/G-Y-F), residues 112-117(including S-D-Y-T-Q-V), and residues 145-147 (Y-K-A). It is believedthat these residues either facilitate or participate in the chemicalreaction, or enable binding of PAPS or the polysaccharide within theactive site. In particular, within residues 43-48, as described aboveand as illustrated in FIG. 1 , the glutamic acid residue at position 43abstracts the proton from the 3-O position of the N-sulfoglucosamineresidue within the polysaccharide, enabling the nucleophilic attack andremoval of the sulfo group from PAPS, whereas His-45 and Asp-48coordinate to stabilize the transition state of the enzyme before thesulfurylated polysaccharide product is released from the active site.

However, as described above, the natural 3OST enzymes are unable tocatalyze the transfer of the sulfate group from an aryl sulfate compoundto a polysaccharide. Without being limited by a particular theory, andas with the NSTs, 2OSTs, and the 6OSTs described above, it is believedthat the binding pocket for PAPS within the active site of the naturalsulfotransferase either does not have a high enough affinity for arylsulfate compounds to facilitate binding and/or that the aryl sulfatecompounds are sterically hindered from entering the active site.Consequently, and in another embodiment, a natural 3OST enzyme can bemutated in several locations within its amino acid sequence to enablebinding of the aryl sulfate compound within the active site and/or tooptimally position the aryl sulfate compound so transfer of the sulfategroup to the polysaccharide can occur.

Accordingly, and in another embodiment, engineered 3OST enzymes that canbe utilized with methods of the present invention can be mutants ofnatural 3OST enzymes within EC 2.8.2.23, including enzymes having theamino acid sequences illustrated in FIGS. 23A-23C. In anotherembodiment, the engineered 3OST enzymes have been engineered torecognize, bind, and react with aryl sulfate compounds as sulfo groupdonors, while retaining the natural enzymes' ability to recognize, bind,and react with any of the HS polysaccharides described above, includingbut not limited to those comprising the structure of Formula X, as sulfogroup acceptors. Without being limited by a particular theory, it isbelieved that because of the mutations inserted into the amino acidsequences of the engineered 3OST enzymes, their sulfotransferaseactivity may comprise the direct transfer of a sulfuryl group from anaryl sulfate compound to the sulfo acceptor polysaccharide, using asimilar mechanism as described in FIG. 1 , above, except that the PAPSis substituted with the aryl sulfate compound. Otherwise, it is believedthat the mutations may cause the sulfotransferase activity to comprise atwo-step process including the hydrolysis of an aryl sulfate compoundand formation of a sulfohistidine intermediate, followed by thenucleophilic attack of the sulfohistidine intermediate by the oxygenatom at the 3-O position of a glucosamine residue, to form a 3-Osulfated product. In another embodiment, the 3-O sulfated HS product isan N,2,3,6-HS product.

In another embodiment, an engineered 3OST enzyme can comprise one ormore mutated amino acid sequence motifs relative to the conserved aminoacid sequence motifs found in natural 3OST enzymes within EC 2.8.2.23,as described above and indicated in the multiple sequence alignment inFIGS. 23A-23C. In another embodiment, each mutated amino acid sequencemotif that is present in the amino acid sequence of the engineeredenzyme comprises at least one amino acid mutation relative to thecorresponding conserved amino acid sequence motif within the natural3OST enzymes. In another embodiment, an engineered 3OST enzyme cancomprise one mutated amino acid sequence motif. In another embodiment,an engineered 3OST enzyme can comprise two mutated amino acid sequencemotifs. In another embodiment, an engineered 3OST enzyme can comprisethree mutated amino acid sequence motifs. In another embodiment, anengineered 3OST enzyme can comprise four mutated amino acid sequencemotifs. In another embodiment, an engineered 3OST enzyme can comprisefive mutated amino acid sequence motifs. In another embodiment, anengineered 3OST enzyme that includes at least one mutated amino acidsequence motif relative to any of the natural 3OST enzymes within EC2.8.2.23 can have an amino acid sequence selected from the groupconsisting of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ IDNO: 57, and SEQ ID NO: 58.

In another embodiment, upon viewing the crystal structure of the mouse3OST1 enzyme within a 3D molecular visualization system (including, as anon-limiting example, the open-source software, PyMOL), the structure ofrelated sequences, such as those of engineered 3OST enzymes that containone or more mutated amino acid sequence motifs relative to the mouse3OST1 (UniProtKB Accession No. O35310) structure, can be modeled forcomparison as illustrated in FIG. 24 . FIG. 24 shows a magnified view ofthe active site of the mouse 3OST enzyme (PDB code: 3UAN) with threeengineered 3OST enzymes, comprising the amino acid sequences of SEQ IDNO: 24, SEQ ID NO: 26, and SEQ ID NO: 28. Adenosine 3′,5′-diphosphate,which is the product of a sulfotransfer reaction in which PAPS is thesulfo donor, and which was co-crystallized with the mouse 3OST, is alsoillustrated within the active site. PNS is also modeled into the activesite of the engineered enzymes, using the consensus solutions ofmolecular dynamics (MD) simulations that designed to calculate theoptimized position and orientation of a ligand within an enzyme activesite adjacent to the polysaccharide binding site (not shown), if suchsolutions are possible. Hydrogen atoms are not shown for clarity.

As illustrated in FIG. 24 , although there are several mutations made toSEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28, relative to the naturalmouse sulfotransferase, the respective protein backbones are in a nearlyidentical location to one another, enabling a one-to-one comparison ofthe active sites. However, when comparing the two active sites, theadenosine 3′,5′-diphosphate product from the natural sulfotransferreaction is adjacent to the lysine residue, whereas the convergentsolutions from the above MD simulations indicate that PNS binding withinthe engineered enzymes is favored on the opposite side of the activesite. Without being limited by a particular theory, it is believed thatthe convergent MD simulation solutions place PNS on the opposite side ofthe active site because there is not enough of an affinity toward PNS inthe same or similar position as PAPS. Yet, despite the apparentdifferences in the binding pocket for PAPS and PNS, engineered 3OSTenzymes comprising the amino acid sequences of SEQ ID NO: 24, SEQ ID NO:26, and SEQ ID NO: 28 all achieved sulfo transfer from an aryl sulfatecompound to the glucosaminyl 3-O position within an N,2,6-HS, asdescribed in the examples below.

Further, the arginine residue corresponding to position 20 of the mouse3OST1, and conserved in all of the other 3OST enzymes illustrated inFIGS. 23A-23C, if present in an engineered 3OST enzyme, would block PNSfrom binding in the position indicated in FIG. 24 . Accordingly, and inanother embodiment, engineered 3OST enzymes that bind PNS can comprise amutation of the active site arginine residue to a glycine residue, whichremoves all steric hindrance for PNS to bind within the binding pocket.As indicated in the amino acid sequences for SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQID NO: 54, the arginine to glycine mutation is at position 21. Asindicated in the amino acid sequences for SEQ ID NO: 56, SEQ ID NO: 57,and SEQ ID NO: 58, the arginine to glycine mutation is at position 99.

Similarly, the next amino acid residue in each of the engineeredenzymes, corresponding to position 22 in the amino acid sequences SEQ IDNO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 53, SEQ ID NO: 54, is mutated to a histidine residue. Withoutbeing limited by a particular theory, it is believed that the mutationto a histidine residue from the conserved lysine residue (correspondingto position 21 in each of the amino acid sequences in FIGS. 23A-23C)facilitates removal of the sulfate group from PNS, using a similarmechanism described by Malojcic, et al., above. As indicated in theamino acid sequences for SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO:58, the lysine to histidine residue is at position 100.

Those skilled in the art would appreciate that engineered 3OST enzymesof any other amino acid sequence, including, but not limited to, thosedisclosed by SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, would exhibit a similarstructure would exhibit similar structural motifs as engineered enzymeshaving the amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 26, and SEQID NO: 28, particularly within the active site. Without being limited bya particular theory, it is also believed that NCS would bind in asimilar position as PNS within the active site of any of the engineeredenzymes, since the structures of the two aryl sulfate compounds are verysimilar, except that the sulfate group is located ortho on the aromaticring relative to the nitro group, rather than para to the nitro group.

In another embodiment, engineered 3OST enzymes that can be utilized inaccordance with methods of the present invention can comprise one ormore mutated amino acid sequence motifs, which can be determined in-partby comparing conserved amino acid sequence motifs indicated in themultiple sequence alignment of FIGS. 23A-23C with the known structure(s)of natural enzymes and/or modeled engineered enzymes, including but notlimited to, as a non-limiting example, enzymes illustrated in FIG. 24 .In another embodiment, mutated amino acid sequence motifs that can becomprised within an engineered 3OST enzyme can be selected from thegroup consisting of (a) G-V-G-H-G-G; (b) H-S-Y-F; (c) S-X₁-X₂-T-H-X₃,wherein X₁ is selected from the group consisting of alanine and leucine;X₂ is selected from the group consisting of tyrosine and glycine, and X₃is selected from the group consisting of methionine and leucine; and (d)Y-X₄-G, wherein X₄ is selected from the group consisting of valine andthreonine; including any combination thereof. Each of the mutated aminoacid sequence motifs corresponds with a conserved amino acid motifindicated in FIGS. 23A-23C above: the mutated amino acid sequence motifG-V-G-H-G-G corresponds to the conserved amino acid sequence motifG-V-R-K-G-G; the mutated amino acid sequence motif H-S-Y-F correspondsto the conserved amino acid sequence motif P-A/G-Y-F; the mutated aminoacid sequence motif S-X₁-X₂-T-H-X₃ corresponds to the conserved aminoacid sequence motif S-D-Y-T-Q-V; and the mutated amino acid sequencemotif Y-X₄-G corresponds to the conserved amino acid sequence motifY-K-A. In another embodiment, an engineered 3OST enzyme comprising eachof the mutated amino acid sequence motifs above can be selected from thegroup consisting of: SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ IDNO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQID NO: 57, and SEQ ID NO: 58.

In another embodiment, each of the mutated amino acid sequence motifscan comprise at least one mutation that is made relative to theconserved amino acids found in the natural 3OST enzymes within EC2.8.2.23. In another embodiment, mutated amino acid sequence motif (a)contains an R-K to G-H mutation, relative to the conserved amino acidsequence motif, G-V-R-K-G-G. In another embodiment, mutated amino acidsequence motif (b) contains a P-A/G to an H-S mutation relative to theconserved amino acid sequence motif, P-A/G-Y-F. In another embodiment,in addition to potential mutations made at the X₁, X₂, and X₃ positions,mutated amino acid sequence motif (c) comprises a Q to H mutation,relative to the conserved amino acid sequence motif, S-D-Y-T-Q-V. Inanother embodiment, in addition to a mutation at the X₄ position,mutated amino acid sequence motif (d) comprises an A to G mutation,relative to the conserved amino acid sequence motif, Y-K-A.

In another embodiment, X₁ is alanine, X₂ is tyrosine; X₃ is methionine,and X₄ is valine or threonine. In other embodiments, X₁ is leucine, X₂is glycine, X₃ is leucine, and X₄ is threonine. Without being limited toanother theory, it is believed that one or more of the mutationscomprised within mutated amino acid sequence motifs (b), (c), and (d)play a role in stabilizing the transition state of the enzyme during thechemical reaction, or in increasing the affinity of aryl sulfatecompounds to the active site, including by reducing the size of thebinding pocket, increasing the hydrophobicity of the pocket, and/orcreating π-π interactions with the aromatic moieties of the aryl sulfatecompounds.

Furthermore, the amino acid sequences (SEQ ID NO: 24, SEQ ID NO: 26, SEQID NO: 28) of three engineered 3OST enzymes, which have beenexperimentally determined to be active with aryl sulfate compounds assulfo group donors (see Example 5 below) can be compared with the aminoacid sequence of the first isoform of the human 3OST enzyme (entrysp|O14792|HS3S1_HUMAN) in a multiple sequence alignment to determine ifthere are relationships between mutations among each of the enzymes. Aperiod within the amino acid sequence of an engineered enzyme indicatesidentity at a particular position with the human 3OST enzyme. As shownin FIG. 25 , the sequence alignment demonstrates that while over 90% ofthe amino acid residues within the three sulfotransferase sequences areidentical, there are several positions in which multiple amino acids canbe chosen. Without being limited by a particular theory, these enzymeshave a similar relationship with each other as the 3OST enzymes thatcomprise EC 2.8.2.23. As a result, and in another embodiment, anengineered 3OST enzyme comprising an amino acid sequence in whichmultiple amino acids can be chosen at defined positions is disclosed asSEQ ID NO: 51. Positions at which the identity of an amino acid can bechosen from a selection of possible residues are denoted in terms “Xaa,”“Xn,” or “position n,” where n refers to the residue position.

In another embodiment, within an engineered 3OST enzyme comprising theamino acid sequence of SEQ ID NO: 51, the amino acid residue at position114 is alanine and the amino acid residue at position 118 is methionine.In further embodiments, the amino acid residue at position 147 isselected from the group consisting of valine and threonine.

In another embodiment, within an engineered 3OST enzyme comprising theamino acid sequence of SEQ ID NO: 51, the amino acid residue at position114 is leucine, the amino acid residue at position 118 is leucine, andthe amino acid residue at position 121 is valine. In furtherembodiments, the amino acid residue at position 115 is glycine. In evenfurther embodiments, the amino acid residue at position 147 isthreonine.

In another embodiment, within an engineered 3OST enzyme comprising theamino acid sequence of SEQ ID NO: 51, the amino acid sequence canoptionally include one or more mutations at residue positions notspecified by an “Xn” or “Xaa,” so long as any such mutations do noteliminate the 3OST and/or aryl sulfate-dependent activity of the enzyme.In another embodiment, such mutations not eliminating arylsulfate-dependent activity at positions not specified by an “Xn” or“Xaa” can include substitutions, deletions, and/or additions.

Accordingly, in another embodiment, an engineered 3OST enzyme utilizedin accordance with any of the methods of the present invention cancomprise an amino acid sequence selected from the group consisting ofSEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 51, SEQ ID NO:52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, and SEQID NO: 58. In another embodiment, any of the above engineered 6OSTenzymes react with an aryl sulfate compound, instead of PAPS, as a sulfogroup donor. In further embodiments, the aryl sulfate compound isselected from the group consisting of PNS, MUS, 7-hydroxycoumarinsulfate, phenyl sulfate, 4-acetylphenyl sulfate, indoxyl sulfate,1-naphthyl sulfate, 2-naphthyl sulfate, and NCS. In some even furtherembodiments, the aryl sulfate compound is PNS. In other even furtherembodiments, the aryl sulfate compound is NCS.

In Vitro Synthesis of Sulfated Polysaccharides

As described above, natural sulfotransferases that recognize, bind, andreact with heparosan-based polysaccharides as sulfo group acceptors havethe ability to produce a wide range of sulfated polysaccharide productsin vivo, including heparin (see Desai, U. R., et al., (1998) J. Biol.Chem. 273 (13):7478-7487). The medical use of heparin has been welldocumented for decades including, but are not limited to, inactivationof Factor IIa (thrombin) and/or Factor Xa, two proteins that are vitalin the blood-clotting cascade. In particular, when heparin binds toantithrombin (AT), it causes a conformational change in the enzyme thatenables the formation of a ternary complex between the polysaccharide,AT, and either thrombin or Factor Xa (see Li, W., et al., (2004) Nat.Struct. Mol. Biol. 11 (9):857-862, the disclosure of which isincorporated by reference in its entirety). In order to bind with AT andinduce its conformational change, polysaccharides within the heparincomposition must have a specific five-residue AT-recognition sequence,which is identical to the structure of Formula I, described above.

While anticoagulation can be induced by binding antithrombin with anoligosaccharide consisting only of the AT-recognition sequence, there istypically an enhanced inhibition of blood clotting when thepolysaccharide comprises more than five sugar residues (see Grey, E., etal., (2008) Thromb. Haemost. 99:807-818, the disclosure of which isincorporated by reference in its entirety). As reported by Grey, et al,a secondary binding interaction can be formed between the N,2,3,6-HSpolysaccharide and thrombin when the polysaccharide comprises at leastthirteen sugar residues on either side of the AT-recognition sequence toact as a “bridge” that facilitates binding to thrombin while also beingbound to AT. As a result, heparin polysaccharides typically require aminimum of eighteen sugar residues in order to potentially form theternary complex between itself, AT, and thrombin. However, and withoutbeing limited by a particular theory, it is believed that because thedistribution of the AT-recognition sequence within a particularpolysaccharide molecule is random, some polysaccharides between eighteenand thirty-one sugar residues can theoretically comprise anAT-recognition sequence toward the center of the molecule that does nothave thirteen adjacent sugar residues on either side. Consequently,N,2,3,6-HS polysaccharides typically must comprise at least thirty-twosugar residues to ensure that the thirteen residue “bridge” adjacent tothe AT-recognition sequence can be formed, no matter where theAT-recognition sequence is within the molecule.

As described above, the hallmark of nearly all sulfotransferases,whether they are utilized in either in vitro or an in vivo sulfotransferreaction, is that they universally and exclusively recognize PAPS as thesulfo group donor, as described in U.S. Pat. Nos. 5,541,095, 5,817,487,5,834,282, 6,861,254, 8,771,995, 9,951,149, and U.S. Pat. Pubs.2009/0035787, 2013/0296540, and 2016/0122446, the disclosures of whichare incorporated by reference in their entireties. These includesulfotransferases in which a polysaccharide is a sulfo group acceptor,particularly HS sulfotransferases that take part in the production ofanticoagulant and non-anticoagulant N,2,3,6-HS products. Currently,because PAPS is expensive and unstable in solution, the most convenientand economically feasible method to obtain anticoagulant N,2,3,6-HSpolysaccharides in large quantities is to isolate them from animalsources, particularly pigs and cattle, rather than to synthesize them invitro, even when a coupled, enzymatic PAPS regeneration system (see U.S.Pat. No. 6,255,088, above) is employed. Without being limited by aparticular theory, utilizing any of the engineered arylsulfate-dependent sulfotransferases described above to catalyze one ormore of the sulfotransfer reactions in the production of N,2,3,6-HSpolysaccharides can reduce the industry's reliance on using PAPS as asulfo group donor, and if an engineered aryl sulfate-dependentsulfotransferase is utilized in all of the enzymatic sulfotransfersteps, the need to use PAPS can be obviated entirely.

Accordingly, methods for synthesizing an N,2,3,6-HS product can compriseany combination of natural or engineered sulfotransferase enzymes, solong as at least one of the reactions comprises an engineered arylsulfate-dependent sulfotransferase enzyme and an aryl sulfate compound.In some embodiments, methods for synthesizing an N,2,3,6-HS product cancomprise the following steps: (a) providing a starting polysaccharidereaction mixture comprising N-deacetylated heparosan; (b) combining thestarting polysaccharide reaction mixture with a reaction mixturecomprising a sulfo group donor and a first sulfotransferase enzymeselected from the group consisting of an NST enzyme, a 2OST enzyme, anda 6OST enzyme, to form a first sulfated polysaccharide; (c) combiningthe first sulfated polysaccharide with a reaction mixture comprising asulfo group donor and a second sulfotransferase enzyme, wherein thesecond sulfotransferase enzyme is one of the two enzymes that were notselected in step (b), to form a second sulfated polysaccharide; (d)combining the second sulfated polysaccharide with a reaction mixturecomprising a sulfo group donor and a third sulfotransferase enzyme,wherein the third sulfotransferase enzyme is the enzyme that was notselected in step (b) or step (c), to form a third sulfatedpolysaccharide; and (e) combining the third sulfated polysaccharide witha reaction mixture comprising a sulfo group donor and a 3OST enzyme, toform the N,2,3,6-HS product. Reaction mixtures that do not comprise anengineered sulfotransferase enzyme can comprise PAPS and a natural HSsulfotransferase enzyme that possesses biological activity with PAPS asthe sulfo group donor. In another embodiment, the reaction mixture thatcomprises the 2OST enzyme further comprises a glucuronyl C₅-epimeraseenzyme.

In another embodiment, when the NST enzyme is an engineered enzyme, theenzyme can comprise an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQID NO: 40. In another embodiment, when the 2OST enzyme is an engineeredenzyme, the enzyme can comprise an amino acid sequence selected from thegroup consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 41, and SEQID NO: 42. In another embodiment, when the 6OST enzyme is an engineeredenzyme, the enzyme can comprise an amino acid sequence selected from thegroup consisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ IDNO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60,and SEQ ID NO: 61. In another embodiment, when the 3OST enzyme is anengineered enzyme, the enzyme can comprise an amino acid sequenceselected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 26, SEQID NO: 28, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58.

In another embodiment, the NST enzyme is the first sulfotransferaseenzyme, the 2OST enzyme is the second sulfotransferase enzyme, and the6OST enzyme is the third sulfotransferase enzyme.

In another embodiment, aryl sulfate compounds used as sulfo group donorscan be selected from the group consisting of PNS, MUS, 7-hydroxycoumarinsulfate, phenyl sulfate, 4-acetylphenyl sulfate, indoxyl sulfate,1-naphthyl sulfate, 2-naphthyl sulfate, and NCS. In even furtherembodiments, the aryl sulfate compound for an engineeredsulfotransferase is PNS. In other even further embodiments, the arylsulfate compound for an engineered sulfotransferase is NCS.

In another embodiment, the N-deacetylated heparosan within the startingpolysaccharide mixture comprises the structure of Formula II. In anotherembodiment, the third sulfated polysaccharide is an N,2,6-HS product. Inanother embodiment, the N,2,6-HS product comprises the structure ofFormula IX. In another embodiment, the N,2,6-HS product comprises thestructure of Formula X. In another embodiment, the N,2,3,6-HS producthas anticoagulant activity. In another embodiment, the N,2,3,6-HSproduct comprises an AT-recognition sequence comprising the structure ofFormula I. In another embodiment, the N,2,3,6-HS product comprising anAT-recognition sequence comprises N,2,3,6-HS polysaccharides having atleast five sugar residues. In another embodiment, the N,2,3,6-HS productcomprising an AT-recognition sequence comprises N,2,3,6-HSpolysaccharides having at least eight sugar residues. In anotherembodiment, the N,2,3,6-HS product comprising an AT-recognition sequencecomprises N,2,3,6-HS polysaccharides having at least eighteen sugarresidues. In another embodiment, the N,2,3,6-HS product comprising anAT-recognition sequence comprises N,2,3,6-HS polysaccharides having atleast thirty-two sugar residues.

In another embodiment, anticoagulant N,2,3,6-HS polysaccharides producedby methods of the present invention can be characterized by the degreeof inhibitory activity that they have against Factor Xa and thrombin,termed “anti-Xa” activity and “anti-IIa” activity, respectively. Theamount of inhibition induced by anticoagulant polysaccharides is oftenmeasured in International Units per milligram (IU mg⁻¹) and less oftenas International Units per milliliter (IU mL⁻¹). In either case, anInternational Unit is an amount approximately equivalent to the quantityrequired to keep 1-mL of cat's blood fluid for 24 hours at 0° C.Typically, the measurable anti-Xa activity of anticoagulant N,2,3,6-HSpolysaccharides is at least about 1 IU mg⁻¹, including at least about 50IU mg⁻¹, at least 75 IU mg⁻¹, 100 IU mg⁻¹, 150 IU mg⁻¹, 200 IU mg⁻¹, or500 IU mg⁻¹, up to at least about 1,000 IU mg⁻¹, and the measurableanti-IIa activity of anticoagulant N,2,3,6-HS polysaccharides is atleast about 1 IU mg⁻¹, including at least about 10 IU mg⁻¹, 25 IU mg⁻¹,50 IU mg⁻¹, 100 IU mg⁻¹, 150 IU mg⁻¹, or 180 IU mg⁻¹, up to at leastabout 200 IU mg⁻¹. For anticoagulant N,2,3,6-HS polysaccharides withinheparin that are thirty-two sugar residues or longer, and are able toform the tertiary complex with AT and thrombin, the ratio of anti-Xaactivity to anti-IIa activity is usually close to 1:1, particularly in arange of 0.9:1 to 1.1:1 (see Keire, D. A., et al., (2011) Anal. Bioanal.Chem. 399:581-591, the disclosure of which is incorporated by referencein its entirety). However, as the chain length decreases belowthirty-two sugar residues and anticoagulant N,2,3,6-HS polysaccharidesare not ensured of interacting with thrombin, the anti-Xa to anti-IIaratio can increase, up to at least about 10.0:1, up to at least 100:1.Consequently, in another embodiment, the ratio of anti-Xa activity toanti-IIa activity of the N,2,3,6-HS product is at least 0.5:1, includingat least 0.75:1, 0.9:1, 1:1, 1.1:1, 1.3:1, 1.5:1, 2.0:1, 3.0:1, 4.0:1,5.0:1, 6.0:1, 7.0:1, 8.0:1, 9.0:1, 10.0:1, 20:1, 40:1, 60:1, or 80:1, upto at least 100:1. In another embodiment, the ratio of anti-Xa activityto anti-IIa activity of the N,2,3,6-HS product is less than 100:1,including less than 80:1, 60:1. 40:1, 20:1, 10.0:1, 9.0:1, 8.0:1, 7.0:1,6.0:1, 5.0:1, 4.0:1, 3.0:1, 2.0:1, 1.5:1, 1.3:1, 1.1:1, 0.9:1, or0.75:1, down to less than 0.5:1. In another embodiment, particularlyfrom about 0.9 to about 1.1. In another embodiment, the ratio of anti-Xaactivity to anti-IIa activity of an N,2,3,6-HS product comprisingpolysaccharides having thirty-two or more sugar residues is in a rangefrom 0.5:1 up to 0.75:1, or 0.9:1, or 1:1, or 1.1:1, or 1.3:1, or 1.5:1,or 2.0:1, or 3.0:1, or 4.0:1, or 5.0:1, or 6.0:1, or 7.0:1, or 8.0:1, or9.0:1, or 10.0:1. In another embodiment, the ratio of anti-Xa activityto anti-IIa activity of an N,2,3,6-HS product comprising polysaccharideshaving thirty-two or more sugar residues is in a range from 0.75:1 up to0.9:1, or 1:1, or 1.1:1, or 1.3:1, or 1.5:1, or 2.0:1, or 3.0:1, or4.0:1, or 5.0:1, or 6.0:1, or 7.0:1, or 8.0:1, or 9.0:1, or 10.0:1. Inanother embodiment, the ratio of anti-Xa activity to anti-IIa activityof an N,2,3,6-HS product comprising polysaccharides having thirty-two ormore sugar residues is in a range from 0.9:1 up to 1:1, or 1.1:1, or1.3:1, or 1.5:1, or 2.0:1, or 3.0:1, or 4.0:1, or 5.0:1, or 6.0:1, or7.0:1, or 8.0:1, or 9.0:1, or 10.0:1. In another embodiment, the ratioof anti-Xa activity to anti-IIa activity of an N,2,3,6-HS productcomprising polysaccharides having thirty-two or more sugar residues isin any range listed above between and inclusive of 0.5:1 and 10.0:1. Insome preferred embodiments, the ratio of anti-Xa activity to anti-IIaactivity of an N,2,3,6-HS product comprising polysaccharides havingthirty-two or more sugar residues is in a range from 0.9:1 up to 1:1.

Similarly, all polysaccharide mixtures, including N,2,3,6-HS productmixtures, can be characterized by their weight-average molecular weight(M _(w)). Because substantially all of the heparins either isolated fromanimal sources or synthesized in vitro are obtained as a polydispersemixture of polysaccharides with different chain lengths and degrees ofsulfation, expressing the average molecular weight as a weight average,rather than a number average (i.e. a true arithmetic mean (M _(n)), isoften the most advantageous because it accounts for the effect largermolecules have on anticoagulation. The M _(w) of a polysaccharidemixture can be measured experimentally using light scattering methods oranalytical ultracentrifugation (see Mulloy, B., et al., (2014) Anal.Bioanal. Chem. 406:4815-4823, the disclosure of which is incorporated byreference in its entirety). However, determining the M _(n), typicallyby size exclusion chromatography, can still be useful because the ratiobetween M _(w) and M _(n) can provide valuable insight into the amountof polydispersity in a particular polysaccharide sample.

In particular, heparin is generally divided into multiple classes basedon their average molecular weights, particularly their M _(w). Samplesof low-molecular weight heparin (LMWH) typically have an M _(w) of lessthan 8,000 Da, in which more than 60% of all of the polysaccharidemolecules within the sample have an actual molecular weight of less than8,000 Da (see Linhardt, R. J. and Gunay, N. S., (1999) Seminars inThrombosis and Hemostasis 25 (Suppl. 3):5-16, the disclosure of which isincorporated by reference in its entirety). LMWH is typically preparedby chemically or enzymatically modifying animal-sourced unfractionatedheparin or API heparin. Unfractionated heparin typically has an M _(w)of greater than 8,000 Da. To be approved for use in medical treatments,API heparin has strict molecular weight guidelines that must be met,namely: (1) the proportion of polysaccharides within the compositionhaving a molecular weight over 24,000 Da is not more than 20%; (2) the M_(w) of the composition itself is between 15,000 Da and 19,000 Da; and(3) the ratio of the number of polysaccharides within the compositionhaving a molecular weight between 8,000 Da and 16,000 Da relative to thenumber of polysaccharides within the composition having a molecularweight between 16,000 Da and 24,000 Da is not less than 1.0:1 (seeMulloy, B., et al., above).

Thus, in another embodiment, the N,2,3,6-HS product that is synthesizedaccording to methods of the present invention can comprise a pluralityof N,2,3,6-HS polysaccharides, and can have one or more molecular weightproperties that are identical to API heparin. In another embodiment, theN,2,3,6-HS product has an M _(w) of at least 1,000 Da, including atleast 2,000 Da, 3,000 Da, 4,000 Da, 5,000 Da, 6,000 Da, 7,000 Da, 8,000Da, 9,000 Da, 10,000 Da, 11,000 Da, 12,000 Da, 13,000 Da, 14,000 Da,15,000 Da, 16,000 Da, 17,000 Da, 18,000 Da, 19,000 Da, 20,000 Da, 21,000Da, 22,000 Da, 23,000 Da, or 24,000 Da, up to at least 50,000 Da. Inanother embodiment, the N,2,3,6-HS product has an M _(w) of less than50,000 Da, including less than 24,000 Da, 23,000 Da, 22,000 Da, 21,000Da, 20,000 Da, 19,000 Da, 18,000 Da, 17,000 Da, 16,000 Da, 15,000 Da,14,000 Da, 13,000 Da, 12,000 Da, 11,000 Da, 10,000 Da, 9,000 Da, 8,000Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, or 3,000 Da, down to lessthan 2,000 Da. In another embodiment, the N,2,3,6-HS product has an M_(w) in a range from 1,000 up to 2,000 Da, or 3,000 Da, or 4,000 Da, or5,000 Da, or 6,000 Da, or 7,000 Da, or 8,000 Da, or 9,000 Da, or 10,000Da, or 11,000 Da, or 12,000 Da, or 13,000 Da, or 14,000 Da, or 15,000Da, or 16,000 Da, or 17,000 Da, or 18,000 Da, or 19,000 Da, or 20,000Da, or 21,000 Da, 22,000 Da, or 23,000 Da, or 24,000 Da. In anotherembodiment, the N,2,3,6-HS product has an M _(w) in a range from 2,000Da up to 3,000 Da, or 4,000 Da, or 5,000 Da, or 6,000 Da, or 7,000 Da,or 8,000 Da, or 9,000 Da, or 10,000 Da, or 11,000 Da, or 12,000 Da, or13,000 Da, or 14,000 Da, or 15,000 Da, or 16,000 Da, or 17,000 Da, or18,000 Da, or 19,000 Da, or 20,000 Da, or 21,000 Da, 22,000 Da, or23,000 Da, or 24,000 Da. In another embodiment, the N,2,3,6-HS productis unfractionated after being produced. In another embodiment, theunfractionated N,2,3,6-HS product has an M _(w) in a range from 8,000 Daup to 9,000 Da, or 10,000 Da, or 11,000 Da, or 12,000 Da, or 13,000 Da,or 14,000 Da, or 15,000 Da, or 16,000 Da, or 17,000 Da, or 18,000 Da, or19,000 Da, or 20,000 Da, or 21,000 Da, 22,000 Da, or 23,000 Da, or24,000 Da. In another embodiment, the anticoagulant N,2,3,6-HS productis an LMW-HS product. In another embodiment, the anticoagulant LMW-HSproduct has an M _(w) in a range from 2,000 Da up to 3,000 Da, or 4,000Da, or 5,000 Da, or 6,000 Da, or 7,000 Da, or 8,000 Da. In anotherembodiment, the anticoagulant N,2,3,6-HS product has an M _(w) in arange from 15,000 Da up to 16,000 Da, or 17,000 Da, or 18,000 Da, or19,000 Da. In another embodiment, the N,2,3,6-HS product can have an M_(w) in any range listed above between and inclusive of 1,000 Da and24,000 Da, and preferably in any range listed above between andinclusive of 15,000 Da and about 19,000 Da.

In another embodiment, less than 50%, including less than 45%, 40%, 35%,30%, 25%, 20%, 15%, 10%, 5%, 3%, or 2%, down to less than 1% of theN,2,3,6-HS polysaccharides within the N,2,3,6-HS product have amolecular weight greater than 24,000 Da. In some preferred embodiments,less than or equal to 20% of the N,2,3,6-HS polysaccharides within theN,2,3,6-HS product have a molecular weight greater than 24,000 Da. Inanother embodiment, when less than or equal to 20% of the N,2,3,6-HSpolysaccharides within the N,2,3,6-HS product have a molecular weightgreater than 24,000 Da, the N,2,3,6-HS product can have an M _(w) in anyrange listed above between and inclusive of 1,000 Da and 24,000 Da, andpreferably in any range listed above between and inclusive of 15,000 Daand about 19,000 Da.

In another embodiment, the relative amount of N,2,3,6-HS polysaccharideshaving a molecular weight between 8,000 Da and 16,000 Da within anN,2,3,6-HS product can be compared as a ratio with the relative amountof N,2,3,6-HS polysaccharides having a molecular weight between 16,000Da and 24,000 Da within the same N,2,3,6-HS product. In anotherembodiment, the ratio of the number of polysaccharides within thecomposition having a molecular weight between 8,000 Da and 16,000 Darelative to the number of polysaccharides within the composition havinga molecular weight between 16,000 Da and 24,000 Da is not less than0.5:1, including not less than 0.75:1, 0.9:1, 1.0:1, 1.1:1, 1.3:1, or1.5:1, up to not less than 2.0:1, and preferably not less than 1.0:1. Inanother embodiment, N,2,3,6-HS products in which the ratio of the numberof polysaccharides within the composition having a molecular weightbetween 8,000 Da and 16,000 Da relative to the number of polysaccharideswithin the composition having a molecular weight between 16,000 Da and24,000 Da is not less than 1.0:1 can also have an M _(w) in any rangelisted above between and inclusive of 1,000 Da and 24,000 Da, andpreferably in any range listed above between and inclusive of 15,000 Daand about 19,000 Da, in which less than or equal to 20% of theN,2,3,6-HS polysaccharides within the N,2,3,6-HS product have amolecular weight greater than 24,000 Da.

In another embodiment, N,2,3,6-HS products prepared by any of themethods of the present invention can satisfy any of the benchmarkrequirements determined by the USP for API heparin, including but notlimited to composition, purity, activity, and/or molecular weight. Inanother embodiment, the anticoagulant N,2,3,6-HS product can possess anyof the properties selected from the group consisting of: an anti-IIaactivity of not less than 180 IU mg⁻¹; an anti-Xa activity of not lessthan 180 IU mg⁻¹; a ratio of anti-Xa to anti-IIa activity in a range of0.9:1 up to 1.1:1, preferably 1:1; an M _(w) of in a range of 15,000 Daup to 19,000 Da; not more than 20% of the polysaccharides having amolecular weight greater than 24,000 Da; and the ratio ofpolysaccharides within the composition having a molecular weight between8,000 Da and 16,000 Da relative to the number of polysaccharides withinthe composition having a molecular weight between 16,000 Da and 24,000Da is not less than 1.0:1; including any combination thereof. In anotherembodiment, anticoagulant N,2,3,6-HS products prepared by any of themethods of the present invention can possess all of the followinganticoagulant activity and molecular weight properties: an anti-IIaactivity of not less than 180 IU mg⁻¹; an anti-Xa activity of not lessthan 180 IU mg⁻¹; a ratio of anti-Xa to anti-IIa activity in a range of0.9:1 up to 1.1:1, preferably 1:1; an M _(w) of in a range of 15,000 Daup to 19,000 Da; not more than 20% of the polysaccharides having amolecular weight greater than 24,000 Da; and the ratio ofpolysaccharides within the composition having a molecular weight between8,000 Da and 16,000 Da relative to the number of polysaccharides withinthe composition having a molecular weight between 16,000 Da and 24,000Da is not less than 1.0:1. In another embodiment, anticoagulantN,2,3,6-HS products prepared by any of the methods of the presentinvention have a substantially equivalent anticoagulant activity andmolecular weight properties relative to API heparin (CAS No: 9041-08-1),which is widely commercially-available.

In another embodiment, anticoagulant N,2,3,6-HS products can satisfybenchmark requirements determined by the USP for API heparin with regardto product purity, particularly purity from other sulfatedpolysaccharides, including but not limited to chondroitin sulfate. Inparticular, over-sulfated chondroitin sulfate (OSCS) was determined tobe the source of contamination within pharmaceutical heparincompositions that caused hundreds of deaths worldwide in 2007 and 2008.In another embodiment, and without being limited by a particular theory,preparations of the N,2,3,6-HS product formed by any of the methods ofthe present invention can be prepared substantially or completely freefrom chondroitin sulfate, particularly OSCS, because it is believed thatthe N-deacetylated heparosan starting material, which can eitherobtained commercially or after modifying heparosan isolated frombacteria (described in further detail below), itself is free ofchondroitin sulfate.

In another embodiment, in order to arrive at N,2,3,6-HS products thatmeet any of the USP molecular weight benchmarks for API heparin, themolecular weight of any of the polysaccharides utilized as sulfo groupacceptors can be controlled. In a non-limiting example, and in anotherembodiment, the molecular weight properties of the heparosan-basedpolysaccharides used as starting materials can be controlled bychemically modifying heparosan until a target set of molecular weightproperties is reached. As described below, heparosan can be obtainedfrom commercial sources or isolated from bacterial or eukaryoticsources.

In particular, heparosan and other heparosan-based polysaccharides suchas heparin are found in several forms of life and have several differentfunctions. In eukaryotes, they operate as sulfo acceptors and/orprecursors in the formation of heparan sulfate and heparin. Heparosanand heparosan-based polysaccharides can also be found within bacteria asa capsule that regulates cell entry by metabolites and other exogenousmaterials. Such bacteria, include, but are not limited to Pasteurellamultocida and Escherichia coli (E. coli). In some embodiments, heparosancan be extracted and purified from E. coli, particularly K5 strain of E.coli, as a polydisperse mixture of polysaccharide molecules havingvarying molecular weights. Procedures for isolating heparosan from theK5 strain of E. coli are discussed and provided in Wang, Z., et al.,(2010) Biotechnol. Bioeng. 107 (6):964-973, the disclosure of which isincorporated by reference in its entirety; see also DeAngelis, P. L.(2015) Expert Opinion on Drug Delivery 12 (3):349-352; Ly, M., et al.,(2010) Anal. Bioanal. Chem. 399:737-745; and Zhang, C., et al., (2012)Metabolic Engineering 14:521-527, the disclosures of which are alsoincorporated in their entireties. However, because substantially all ofthe heparosan isolated from bacteria, including E. coli, isN-acetylated, it cannot be used directly as a sulfo acceptor for any ofthe sulfotransferases described herein and utilized in accordance withthe methods of the present invention. As a result, heparosan must be atleast partially N-deacetylated before it can be utilized as a sulfogroup acceptor.

As a result, and in another embodiment, heparosan can be at leastpartially N-deacetylated by treating it with a base, particularlylithium hydroxide or sodium hydroxide (see Wang, Z., et al., (2011)Appl. Microbiol. Biotechnol. 91 (1):91-99, the disclosure of which isincorporated by reference in its entirety; see also PCT publicationPCT/US2012/026081, the disclosure of which is incorporated by referencein its entirety). In another embodiment, the base is sodium hydroxide.Depending on the degree of N-deacetylation desired, the concentration ofthe heparosan, and the concentration of the base, one skilled in the artcan determine how long to incubate heparosan with the base according tothe procedures described in Wang, et al., (2011), above.

In another embodiment, heparosan can be incubated with a base,preferably sodium hydroxide, until a desired amount of N-acetylatedglucosamine residues remains within the N-deacetylated product. Inanother embodiment, N-acetyl glucosamine residues can comprise less than60%, including less than 30%, 20%, 18%, 16%, 14%, 12%, or 10%, down toless than 5%, and preferably in a range from 12% and up to 18%, of theglucosamine residues within the N-deacetylated heparosan product. Inanother embodiment, the N-acetyl glucosamine can comprise about 15% ofthe glucosamine residues within the N-deacetylated heparosan product.

Additionally, and without being limited by a particular theory, it isbelieved that in addition to N-deacetylating glucosamine residues, thereaction between heparosan and a base can simultaneously depolymerizethe heparosan polysaccharides and reduce their molecular weight, whichcan in turn reduce the M _(w) of the N-deacetylated heparosancomposition. Typically, heparosan polysaccharides isolated frombacteria, including but not limited to E. coli, have a molecular weightranging from about 3,000 Da to about 150,000 Da, and compositions ofisolated heparosan can have a M _(w) in the range of about 25,000 Da upto about 50,000 Da (see Ly, M., et al. and Wang, et al., (2011), above).In another embodiment, and independent from its starting M _(w) andoverall molecular weight properties, a heparosan composition eitherobtained from commercial sources or isolated from bacteria, includingbut not limited to E. coli, can be treated with a base, preferablysodium hydroxide, for a time sufficient to reduce the M _(w) of theN-deacetylated heparosan product to a target or desired level. Inanother embodiment, the depolymerized, N-deacetylated heparosan producthas an M _(w) of at least 1,000 Da, including at least 2,000 Da, 4,000Da, 6,000 Da, 7,000 Da, 8,000 Da, 8,500 Da, 9,000 Da, 9,500 Da, 10,000Da, 10,500 Da, 11,000 Da, 11,500 Da, 12,000 Da, 12,500 Da, 13,000 Da,13,500 Da, 14,000 Da, 15,000 Da, 16,000 Da, or 18,000 Da, up to at least20,000 Da. In another embodiment, the depolymerized, N-deacetylatedheparosan product has an M _(w) of less than 20,000 Da, including lessthan 18,000 Da, 16,000 Da, 15,000 Da, 14,000 Da, 13,500 Da, 13,000 Da,12,500 Da, 12,000 Da, 11,500 Da, 11,000 Da, 10,500 Da, 10,000 Da, 9,500Da, 9,000 Da, 8,500 Da, 8,000 Da, 7,000 Da, 6,000 Da, or 4,000 Da, downto less than 2,000 Da. In another embodiment, the depolymerized,N-deacetylated heparosan product has an M _(w) in a range from 1,000 upto 2,000 Da, or 4,000 Da, or 6,000 Da, or 7,000 Da, or 8,000 Da, or8,500 Da, or 9,000 Da, or 9,500 Da, or 10,000 Da, or 10,500 Da, or11,000 Da, or 11,500 Da, or 12,000 Da, or 12,500 Da, or 13,000 Da, or13,500 Da, or 14,000 Da, or 15,000 Da, or 16,000 Da, or 18,000 Da, or20,000 Da. In another embodiment, the anticoagulant N,2,3,6-HS producthas an M _(w) in a range from 7,000 Da up to 8,000 Da, or 8,500 Da, or9,000 Da, or 9,500 Da, or 10,000 Da, or 10,500 Da, or 11,000 Da, or11,500 Da, or 12,000 Da, or 12,500 Da, or 13,000 Da, or 13,500 Da, or14,000 Da, or 15,000 Da. In another embodiment, the depolymerized,N-deacetylated heparosan product has an M _(w) in a range from 9,000 Daup to 9,500 Da, or 10,000 Da, or 10,500 Da, or 11,000 Da, or 11,500 Da,or 12,000 Da, or 12,500 Da. In another embodiment, the depolymerized,N-deacetylated heparosan product can have an M _(w) in any range listedabove between and inclusive of 1,000 Da and 20,000 Da, and preferably inany range listed above between and inclusive of 9,000 Da and 12,500 Da.

In another embodiment, a heparosan composition can be treated with abase, preferably sodium hydroxide, for a time sufficient to both reducethe M _(w) of the N-deacetylated heparosan product to a target ordesired level, and to attain a desired amount of glucosamine residuesthat remain N-acetylated within the N-deacetylated heparosan product.Methods for providing a starting polysaccharide reaction mixturecomprising N-deacetylated heparosan comprise the following sub-steps:(a) providing a precursor polysaccharide composition comprisingheparosan; and (b) combining the precursor polysaccharide compositionwith a reaction mixture comprising a base, preferably lithium hydroxideor sodium hydroxide, for a time sufficient to N-deacetylate at least oneof the N-acetylated glucosamine residues within the heparosan, formingthe N-deacetylated heparosan composition. In another embodiment, theN-deacetylated heparosan product can have an M _(w) in any range listedabove between and inclusive of 1,000 Da and 20,000 Da, simultaneouslywith having less than 60% of the glucosamine residues within theN-deacetylated heparosan product present as N-acetylglucosamineresidues. In another embodiment, the N-deacetylated heparosan productcan have an M _(w) in any range listed above between and inclusive of9,000 Da and 12,500 Da, in which from 12% and up to 18% of theglucosamine residues within the N-deacetylated heparosan product areN-acetylated. The preparation of N-deacetylated heparosan having suchmolecular weight properties and N-acetyl content is described in detailin Wang, et al., (2011), above. In another embodiment, the timesufficient to react a heparosan with a base, preferably sodiumhydroxide, to form an N-deacetylated heparosan product having an M _(w)in a range between 9,000 Da and 12,500 Da, as well as an N-acetylglucosamine content in a range from 12% and up to 18%, can be at least 1hour, including at least 2, 4, 6, 8, 10, 12, or 18 hours, and up to atleast 24 hours, depending on the molecular weight properties andconcentration of the heparosan starting material, and the identity andconcentration of the base used to carry out the reaction.

In another embodiment, N-deacetylated heparosan can be combined with anN-sulfation agent within a reaction mixture to form N-sulfatedheparosan. As described above, and in another embodiment, theN-sulfation agent can comprise any of the natural or engineered NSTenzymes described above. In another embodiment, when the N-sulfationagent is a natural NST, the reaction mixture can also comprise PAPS as asulfo group donor. In another embodiment, when the N-sulfation agent isan engineered NST, the reaction mixture can also comprise an arylsulfate compound, preferably PNS or NCS, as a sulfo group donor.

In another embodiment, N-deacetylated heparosan can be chemicallyN-sulfated, rather than being enzymatically N-sulfated. In anotherembodiment, the N-sulfation agent is a chemical agent, preferably sulfurtrioxide and/or one or more sulfur-trioxide containing compounds oradducts. Chemical N-sulfation of glucosamine residues withinpolysaccharides using sulfur trioxide is commonly known in the art (seeLloyd, A. G., et al., (1971) Biochem. Pharmacol. 20 (3):637-648;Nadkarni, V. D., et al., (1996) Carbohydrate Research 290:87-96;Kuberan, B., et al., (2003) J Biol. Chem. 278 (52):52613-52621; Zhang,Z., et al., (2008) J. Am. Chem. Soc. 130 (39):12998-13007; and Wang, etal., (2011), above; see also U.S. Pat. No. 6,991,183 and U.S. Pat. Pub.2008/020789, the disclosures of which are incorporated by reference intheir entireties). Sulfur trioxide complexes are generally mild enoughbases to enable the selected N-sulfation of polysaccharides withoutcausing depolymerization, unlike sodium hydroxide (see Gilbert, E. E.,(1962) Chem. Rev. 62 (6):549-589). Non-limiting examples of sulfurtrioxide-containing complexes include sulfur dioxide-pyridine, sulfurdioxide-dioxane, sulfur dioxide-trimethylamine, sulfurdioxide-triethylamine, sulfur dioxide-dimethylaniline, sulfurdioxide-thioxane, sulfur dioxide-Bis(2-chloroethyl) ether, sulfurdioxide-2-methylpyridine, sulfur dioxide-quinoline, or sulfurdioxide-dimethylformamide. In another embodiment, the N-sulfation agentcomprises a sulfur trioxide-containing adduct selected from the groupconsisting of a sulfur trioxide-trimethylamine adduct and a sulfurtrioxide-pyridine adduct. In another embodiment, the N-sulfation agentcomprises a sulfur trioxide-trimethylamine adduct.

In another embodiment, N-sulfation, particularly chemical N-sulfation,can comprise the first sulfation step, with respect to N-deacetylatedheparosan. Subsequently, after the N-deacetylated heparosan is eitherenzymatically or chemically N-sulfated, the N-sulfated heparosan canthen be further sulfated using a 2OST, 6OST, and 3OST. In embodiments inwhich an anticoagulant N,2,3,6-HS product is formed, enzymatic sulfationsteps occur in the order of 2-O, 6-O, and 3-O sulfation. As describedabove, the 3OST enzyme, and preferably all of the sulfotransferaseenzymes, are engineered aryl-sulfate dependent sulfotransferase enzymes,and the reactions are performed in the absence of PAPS. In anotherembodiment, the reaction mixture comprising the 2OST enzyme furthercomprises a glucuronyl C₅-epimerase enzyme, preferably a glucuronylC₅-epimerase enzyme comprising the amino acid sequence of SEQ ID NO: 29,and more preferably a glucuronyl C₅-epimerase enzyme comprising theamino acid sequence of residues 34-617 of SEQ ID NO: 29. In anotherembodiment, the N,2,3,6-HS product comprises anticoagulant activity. Inanother embodiment, the N,2,3,6-HS product comprises an AT-recognitionsequence comprising the structure of Formula I.

In another embodiment, any of the methods for forming an N,2,3,6-HSproduct described above can be performed sequentially, and each sulfatedpolysaccharide product can be isolated and purified prior to beingtreated with another sulfotransferase in a subsequent step. In anotherembodiment, at least two of the steps can be performed in a single pot,and the sulfated polysaccharide product can be isolated and purifiedfrom that pot before being utilized in a subsequent sulfotransfer step.In another embodiment, one non-limiting combination of sulfotransferreactions that can take place in a single pot includes N-sulfation and2-O sulfation steps, after which the N,2-HS product is isolated andpurified prior to reacting with the 6OST. Without being limited by aparticular theory, the N-sulfated HS product can either be utilized asulfo acceptor for the 2OST enzyme directly and/or the reaction mixturecan comprise any of the glucuronyl C₅-epimerase enzymes described aboveto catalyze the conversion between polysaccharides comprising thestructure of Formula IV and Formula V. However, and in still furtherembodiments, the reaction mixtures and enzymes for any combination ofsulfotransferase reactions can be combined within a single pot,including reaction mixtures and enzymes for all four sulfationreactions, and at least a 2OST, a 6OST, and a 3OST.

In another embodiment, within any of the methods for forming anN,2,3,6-HS product described above, any of the reaction mixturescomprising an engineered sulfotransferase and an aryl sulfate compoundas a sulfo group donor can further comprise one or more reactioncomponents for repopulating the aryl sulfate compound. In anotherembodiment, the one or more reaction components comprise an arylsulfotransferase (ASST) enzyme and a secondary aryl sulfate compound. Innature, aryl sulfotransferase enzymes can catalyze the sulfation ofaromatic compounds to form an aryl sulfate compound. Typically, thesulfo donor itself is an aryl sulfate compound. The reactivity of ASSTenzymes is generally described, for example, in U.S. Pat. Nos. 6,225,088and 8,771,995, as well as Malojcic, et al., above, the disclosures ofwhich are incorporated by reference in their entireties. Without beinglimited by a particular theory, it is believed that including an ASSTand a secondary aryl sulfate compound within a reaction mixturecomprising an engineered sulfotransferase can have the advantage ofreducing potential competitive inhibition of the engineeredsulfotransferase by the desulfated aromatic product, as well asrepopulating the reaction mixture with the sulfo group donor.

In another embodiment, the secondary aryl sulfate compound can be anyaryl sulfate compound, including those described above. In anotherembodiment, the secondary aryl sulfate compound is the same aryl sulfatecompound used as the sulfo group donor for the engineeredsulfotransferase enzyme. In another embodiment, the secondary arylsulfate compound is a different aryl sulfate compound than the one usedas the sulfo group donor for the engineered sulfotransferase enzyme. Asa non-limiting example, and in another embodiment, if the engineeredsulfotransferase has biological activity with NCS as a sulfo groupdonor, then the secondary aryl sulfate compound is PNS. In anothernon-limiting example, and in another embodiment, if the engineeredsulfotransferase has biological activity with PNS as a sulfo groupdonor, then the secondary aryl sulfate compound is NCS.

In another embodiment, the ASST enzyme utilized in conjunction with anyof the above methods to repopulate the sulfo donor aryl sulfate compoundcan be any bacterial enzyme, either isolated from in vivo sources orgenerated recombinantly in vitro, which transfers a sulfo group from anaryl sulfate compound to an aromatic compound. In another embodiment,and in one non-limiting example, the ASST is a recombinant ASST from E.coli, preferably from the E. coli strain CFT073 and having the aminoacid sequence of SEQ ID NO: 55. In another embodiment, an ASST enzyme,preferably an ASST enzyme comprising the amino acid sequence of SEQ IDNO: 55, when coupled to any of the engineered sulfotransferasesdescribed above, can transfer a sulfate group from the secondary arylsulfate compound to the desulfated aromatic compound formed by theengineered sulfotransferase. Without being limited by a particulartheory, it is believed that utilizing the ASST can reduce potentialproduct inhibition by the desulfated aromatic compound, while alsoregenerating the sulfo group donor for subsequent sulfotransferreactions to an HS or heparosan-based polysaccharide.

In another embodiment, and also without being limited by a particulartheory, it is believed that coupling the engineeredsulfotransferase-catalyzed reaction with ASST can provide a furtheradvantage of generating the aryl sulfate sulfo donor directly from anon-sulfated aromatic compound. The reaction mixture for a particularreaction catalyzed by an engineered sulfotransferase can be formulatedto combine a non-sulfated aromatic compound with ASST and a secondaryaryl sulfate compound either prior to or simultaneously with addition ofthe engineered sulfotransferase to the reaction mixture. In anon-limiting example, and in another embodiment, a sulfotransferreaction catalyzed by an engineered sulfotransferase enzyme can beinitiated by combining a non-sulfated aromatic compound, an aryl sulfatecompound, and an ASST in the same reaction mixture as the engineeredsulfotransferase and the polysaccharide sulfo group acceptor. Thereaction between the ASST, the aryl sulfate compound, and thenon-sulfated aromatic compound can generate the sulfo donor aryl sulfatecompound, which can then react with the engineered sulfotransferaseenzyme to transfer the sulfate group to the polysaccharide. In anotherembodiment, the aryl sulfate compound produced by the reaction with theASST enzyme is a different compound than the aryl sulfate compound thatreacts with ASST itself. In a non-limiting example, the non-sulfatedaromatic compound is NCS, and the aryl sulfate compound that reacts withthe ASST is PNS. As NCS is formed by the reaction between PNS and ASST,the sulfo group can then be transferred from the NCS to thepolysaccharide, using the engineered sulfotransferase.

Post-Synthesis Processing of N,2,3,6-HS Products

As described above, API heparin generally adheres to a tightly-regulatedset of molecular weight and activity requirements, whereas LMWHgenerally has an average molecular weight of less than 8,000 Da, inwhich more than 60% of all of the polysaccharide molecules within thesample have an actual molecular weight of less than 8,000 Da (seeLinhardt, R. J. and Gunay, N. S., above). Furthermore, LMWH drugs havetheir own regulated set of composition, purity, molecular weight andactivity requirements in their own right, and are generally preparedfrom unfractionated heparin or API heparin. Accordingly, and in anotherembodiment, N,2,3,6-HS products produced by any of the methods describedabove can be utilized to produce LMW-HS products, using any well-knownmeans in the art. In another embodiment, the N,2,3,6-HS product producedby any of the methods described above and utilized in the synthesis ofan LMW-HS product has anticoagulant activity. In another embodiment, theN,2,3,6-HS product produced by any of the methods described above andutilized in the synthesis of an LMW-HS product has molecular weightand/or anticoagulant activity properties that are identical to APIheparin. In another embodiment, the LMW-HS product synthesized from anN,2,3,6-HS product produced by any of the methods described above alsohas anticoagulant activity. Non-limiting exemplary methods forsynthesizing LMW-HS products from N,2,3,6-HS products are described infurther detail below.

In one non-limiting example, and in another embodiment, N,2,3,6-HSpolysaccharides within an N,2,3,6-HS product mixture that have a lowmolecular weight, particularly a molecular weight less than 15,000 Da,including less than 14,000 Da, 13,000 Da, 12,000 Da, 11,000 Da, 10,000Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, or 3,000Da, down to less than 2,000 can be separated from other N,2,3,6-HSpolysaccharides within the same mixture. In another embodiment,N,2,3,6-HS polysaccharides within an N,2,3,6-HS product mixture can beseparated by electrophoretic mobility using gel electrophoresis. Inanother embodiment, N,2,3,6-HS polysaccharides within an N,2,3,6-HSproduct mixture can be separated by size exclusion chromatography. Inanother embodiment, N,2,3,6-HS polysaccharides within an N,2,3,6-HSproduct mixture can be separated by precipitation with salts of adivalent cation and a weak anion, including but not limited to barium,calcium, magnesium, strontium, copper, nickel, cadmium, zinc, mercury,beryllium, palladium, platinum, iron, and tin salts. In anotherembodiment, the polysaccharides can be separated from highermolecular-weight polysaccharides in bulk, by separating all suchN,2,3,6-HS polysaccharides under 15,000 Da from those above 15,000 Da,as a non-limiting example. In another embodiment, the polysaccharidescan be separated into one or more fractions, such as 10,000 Da to 15,000Da, 5,000 Da to 10,000 Da, and all N,2,3,6-HS polysaccharides under5,000 Da, as another non-limiting example.

In another embodiment, N,2,3,6-HS polysaccharide product mixtures havingan average molecular weight less than 8,000 Da can be utilized as LMW-HSproducts directly. In other embodiments, N,2,3,6-HS polysaccharideproduct mixtures having an average molecular weight less than 8,000 Dacan be combined with other glycosaminoglycans (GAGs) to form HS-GAGmixtures. Although an advantage of several of the methods above,particularly methods in which the heparosan starting material isisolated and purified from E. coli, includes the ability to synthesizeN,2,3,6-HS products that are free from chondroitin sulfate, dermatansulfate, and other sulfated GAGs, some highly-purified HS-GAG mixturesthat comprise chondroitin sulfate and/or dermatan sulfate have beensuccessfully prescribed to patients in the past because they havebeneficial pharmacological properties relative to heparin, even if theydon't possess as much anticoagulant activity as heparin. Non-limitingexamples of HS-GAG mixtures that have been prescribed medically includesulodexide (CAS No: 57821-29-1) and danaparoid (CAS No: 308068-55-5). Inanother embodiment, HS-GAG mixtures formed between an anticoagulantN,2,3,6-HS products synthesized by any of the methods of the presentinvention and one or more GAGs can have anticoagulant activity.

Historically, sulodexide has been extracted from pig intestinal mucosa(see U.S. Pat. No. 3,936,351, herein incorporated by reference in itsentirety), but sulodexide can also be prepared by combining dermatansulfate (CAS No: 24967-94-0) with the “fast-moving” heparin fractionsthat can be separated from heparin using salt precipitation (see Volpi,N., (1993) Carbohydr. Res. 247:263-278), particularly with barium salts.Fast-moving heparin fractions (FM-HS) are deemed “fast-moving” based ontheir electrophoretic mobility relative to heavier, “slow-moving”heparin (SM-HS) that are also formed upon salt precipitation of heparin,and can be purified away from SM-HS, using ultracentrifugation, as anon-limiting example. Additionally, FM-HS fractions have reducedanticoagulant activity and overall sulfation relative to heparin, and arelative molecular mass, M_(r), as determined by high performance sizeexclusion chromatography (HPSEC) of about 8,000 (see Volpi, N., above).However, the mean molecular weight of the FM-HS fraction itself is about7,000 Da (see Coccheri, S. and Mannello, F., (2014) Drug Design,Development, and Therapy 8:49-65).

Further, the FM-HS fractions that are separated from heparin generallyhave similar chemical properties to other LMWH compositions, including alonger half-life and increased oral bioavailability relative to APIheparin. On the other hand, dermatan sulfate generally has minimal to noanticoagulant activity and an average molecular weight of 25 kDa, buthas been shown to inhibit arterial and venous thrombosis, and to provideprotection against vascular wall damage and inflammation as well asaccelerated wound healing. Without being limited by a particular theory,it is believed that the combination of FM-HS and dermatan sulfate withinsulodexide can react in combinatory, and potentially synergistic,fashion. (see Coccheri, S. and Mannello, F., above.)

Thus, in another embodiment, FM-HS fractions are prepared fromanticoagulant N,2,3,6-HS products synthesized by any of the methods ofthe present invention, using engineered aryl sulfate-dependentsulfotransferase enzymes. In another embodiment, the N,2,3,6-HS productprepared using the engineered sulfotransferase enzymes can beprecipitated with divalent-cationic salt, particularly a barium orcalcium salt, using a similar procedure described by Volpi, above. Inanother embodiment, the N,2,3,6-HS product is substantially equivalentto API heparin. Methods for performing a salt precipitation of APIheparin to form and subsequently purify FM-HS are also described in U.S.Pat. Nos. 7,687,479 and 8,609,632, the disclosures of which are hereinincorporated by reference in their entireties. In another embodiment,once the resulting FM-HS fraction is purified, it can be combined withdermatan sulfate to form an HS-GAG mixture. In another embodiment, anyof the methods of the present invention can be utilized to synthesizeFM-HS directly, which can then be combined with dermatan sulfate to forman HS-GAG mixture. In another embodiment, the HS-GAG mixture prepared byeither method can comprise one or more properties that are identical tosulodexide, including but not limited to a composition comprising 80% ofthe FM-HS fraction and 20% of dermatan sulfate (see Lauver, D. A.Lucchesi, B. R., Cardio. Drug Rev. 24 (3-4):214-216), an averagemolecular weight of 7,000 Da, an M_(r) of about 8,000, and/or a sulfateto carboxyl group ratio in the range of 2.0:1 to 2.2:1.

In contrast to sulodexide, the HS-GAG mixture, danaparoid, has beenhistorically prepared from natural HS isolated from porcine sources,rather than API heparin (see U.S. Pat. No. 5,164,377, hereinincorporated by reference in its entirety; see also “Danaparoid Sodium”(2010) European Pharmacopoeia 7.0, 1789-1792). HS polysaccharides, asopposed to heparin, contain disaccharide units that are generally eitherunsulfated or are N-, 2-O, and/or 6-O sulfated. Without being limited bya particular theory, however, it is believed that disaccharide unitscomprising 3-O sulfated glucosamine residues are rare within HS,resulting in a dramatically reduced anticoagulant activity relative toheparin. Accordingly, danaparoid also has a reduced activity relative toheparin, generally having an anti-Xa activity of 11-20 IU mg⁻¹, ananti-IIa activity of less than 1 IU mg⁻¹, and a ratio of anti-Xaactivity to anti-IIa activity of not less than 22:1.

Additionally, upon purifying danaparoid according to the procedures inU.S. Pat. No. 5,164,377, the resulting product contains not only HS, butalso chondroitin sulfate and dermatan sulfate, that have reducedmolecular weights as a result of the addition of a base during theextraction process, similar to the effect of reacting a base withheparosan to reduce the molecular weight. According to the EuropeanPharmacopoeia, the weight-average molecular weight (M _(w)) of all ofthe GAGs within a danaparoid HS-GAG composition suitable to beprescribed to patients is in a range of at least 4,000 Da, up to 7,000Da, and comprise the following size distribution limits: (a)polysaccharide chains comprising an M_(r) of less than 2,000 comprise amaximum of 13% (w/w) of the danaparoid mixture; (b) polysaccharidechains comprising an M_(r) of less than 4,000 comprise a maximum of 39%(w/w) of the danaparoid mixture; (c) polysaccharide chains comprising anM_(r) between 4,000 and 8,000 comprise a minimum of 50% (w/w) of thedanaparoid mixture; (d) polysaccharide chains comprising an M_(r) ofhigher than 8,000 comprise a maximum of 19% (w/w) of the danaparoidmixture; and (e) polysaccharide chains comprising an M_(r) of less than10,000 comprise a maximum of 11% (w/w) of the danaparoid mixture. Withregard to particular composition limits for danaparoid determined by theEuropean Pharmacopoeia, chondroitin sulfate can comprise a maximum of8.5% (w/w) of the danaparoid mixture, and dermatan sulfate can comprisea range from at least 8.0% (w/w) up to 16.0% (w/w) of the danaparoidmixture. As a non-limiting example, the danaparoid composition Orgaran®comprises about 84% (w/w) HS, about 12% (w/w) dermatan sulfate, andabout 4% chondroitin sulfate.

In another embodiment, an HS-GAG mixture comprising an HS productproduced by any of the methods of the present invention using engineeredaryl sulfate-dependent sulfotransferase enzymes, dermatan sulfate, andchondroitin sulfate can be formed that has similar properties todanaparoid (CAS No: 308068-55-5). In another embodiment, the HS productis an N,2,6-HS product. In another embodiment, the HS product is anN,2,3,6-HS product. In another embodiment, the HS product synthesizeddirectly from the reaction has an M _(w) in a range from at least 4,000Da, and up to 8,000 Da, preferably in a range from at least 4,000 Da, upto 7,000 Da. In another embodiment, the HS product has an M _(w) largerthan 8,000 Da, and is prepared for inclusion in a danaparoid-like HS-GAGmixture by subsequently reacting it with a base, similar to methodsdescribed above for depolymerizing heparosan, to reduce its molecularweight. In another embodiment, chondroitin sulfate and dermatan sulfateare reacted with a base to reduce their molecular weight. In anotherembodiment, a composition comprising an HS product produced by any ofthe methods of the present invention, chondroitin sulfate, and dermatansulfate can be filtered using a filtration device. Such filtrationdevices can include, but are not limited to, centrifugal filter unitssuch as an Amicon® Ultra unit (EMD Millipore), or dialysis membranes,either of which have a desired molecular weight cut-off (MWCO). Inanother embodiment, the MWCO for either a centrifugal filter unit ordialysis membrane is 5,500 Da. In another embodiment, the M _(w) for allof the GAGs in the danaparoid HS-GAG mixture is in a range from at least4,000 Da, and up to 8,000 Da, preferably in a range from at least 4,000Da, and up to 7,000 Da, and more preferably in a range from at least5,000 Da, and up to 6,000 Da. In another embodiment, GAGs within thedanaparoid HS-GAG mixture comprise the following size distributionlimits: (a) polysaccharide chains comprising an M_(r) of less than 2,000comprise a maximum of 13% (w/w) of the danaparoid HS-GAG mixture; (b)polysaccharide chains comprising an M_(r) of less than 4,000 comprise amaximum of 39% (w/w) of the danaparoid HS-GAG mixture; (c)polysaccharide chains comprising an M_(r) between 4,000 and 8,000comprise a minimum of 50% (w/w) of the danaparoid HS-GAG mixture; (d)polysaccharide chains comprising an M_(r) of higher than 8,000 comprisea maximum of 19% (w/w) of the danaparoid HS-GAG mixture; and (e)polysaccharide chains comprising an M_(r) of less than 10,000 comprise amaximum of 11% (w/w) of the danaparoid HS-GAG mixture.

In another embodiment, the danaparoid HS-GAG mixture can comprise a GAGcomposition that is either similar or identical to danaparoid (CAS No:308068-55-5). In another embodiment, the composition of the GAGs withinthe danaparoid HS-GAG mixture comprises at least 8% (w/w), up to 16%(w/w), and preferably 12% (w/w) of dermatan sulfate, and less than 8%(w/w), preferably in a range of at least 3% (w/w), up to 5% (w/w), andmore preferably 4% (w/w) of chondroitin sulfate.

In another embodiment, the danaparoid HS-GAG mixture can comprise eithera similar or identical anticoagulant activity to danaparoid. In anotherembodiment, the danaparoid HS-GAG mixture can comprise an anti-Xaactivity of 11-20 IU mg⁻¹, an anti-IIa activity of less than 1 IU mg⁻¹,and/or a ratio of anti-Xa activity to anti-IIa activity of not less than22:1.

In another embodiment, rather than combining HS products, particularlyanticoagulant N,2,3,6-HS products, synthesized according to any of themethods of the present invention to form HS-GAG mixtures, the HSproducts can instead be further modified by one or more subsequentprocesses to depolymerize and/or modify the HS product to form an LMW-HSproduct, as described above. Generally, and in another embodiment, theprocess for forming an LMW-HS from an anticoagulant N,2,3,6-HS productcomprises the following steps: (a) synthesizing an N,2,3,6-HS productaccording to any of the above methods; (b) providing one or moredepolymerization agents; and (c) treating the N,2,3,6-HS product withthe one or more depolymerization agents for a time sufficient todepolymerize at least a portion of the N,2,3,6-HS product, therebyforming the LMW-HS product. Without being limited by a particulartheory, it is believed that the choice in the depolymerization agent candetermine the chemical mechanism for forming the LMW-HS product, as wellas the product(s) structure, anticoagulant activity, and pharmacologicalproperties. Known chemical mechanisms for forming an LMW-HS product frompharmaceutical heparin include, but are not limited to: chemical and/orenzymatic β-elimination reactions; deamination reactions; and oxidationreactions, including combinations thereof.

In another embodiment, an N,2,3,6-HS product, synthesized according toany of the methods of the present invention, can be modified by anenzymatic β-elimination reaction to form an enzymatically-depolymerizedLMW-HS product. Historically, enzymatically-depolymerized LMW-HSproducts have been prepared by incubating pharmaceutical heparin withone or more carbon-oxygen lyase enzymes until the LMW-HS productcomprises a desired chemical structure, average molecular weight,anticoagulant activity, and degree of sulfation. (see “TinzaparinSodium” (2010) European Pharmacopoeia 7.0, 3098; see also Linhardt, R.J. and Gunay, N. S., above). As a result of the reaction with the one ormore carbon-oxygen lyases, the polysaccharide within the heparin bothdepolymerize and develop a characteristic chemical structure,illustrated by Formula XI, below.

As illustrated above in Formula XI, n can be any integer from 1-25.Instead of a glucuronic acid or uronic acid residue, the sugar residueat the non-reducing end of a majority of the enzymatically-depolymerizedLMW-HS polysaccharides within the product is a2-O-sulfo-4-enepyranosulfonic acid. Additionally, each glucosamineresidue at the reducing end is sulfated at the N- and 6-O positions.Optionally, the 3-O position of a glucosamine residue within one or moreof disaccharide units can also be 3-O sulfated. Without being limited bya particular theory, it is believed that at least some of thepolysaccharides within the enzymatically-depolymerized LMW-HS productcomprises 3-O sulfated glucosamine residues, which ultimately leads toleads to its anticoagulant activity.

Further, much like heparin, enzymatically-depolymerized LMWH productsderived from heparin that can be prescribed as anticoagulants mustsatisfy strict purity and property standards. In particular, one suchenzymatically-depolymerized LMWH product, tinzaparin (CAS No: 9041-08-1;ATC code: B01AB10), has a particular set of molecular weight,anticoagulant activity, and sulfation content properties in addition tothe chemical structure of Formula XI above, including: an M _(w) in arange from at least 5,500 Da, and up to 7,500 Da, and characteristically6,500 Da; at least 1.8 and up to 2.5 sulfate groups per disaccharideunit; and an anti-Xa activity of at least 70 IU mg⁻¹ and up to 120 IUmg⁻¹, and/or a ratio of anti-Xa activity to anti-IIa activity of atleast 1.5:1, and up to 2.5:1.

Accordingly, in another embodiment, an N,2,3,6-HS product synthesizedaccording to any of the methods of the present invention described abovecan subsequently be depolymerized by one or more carbon-oxygen lyases toform an enzymatically-depolymerized LMW-HS product. In anotherembodiment, the enzymatically-depolymerized LMW-HS product comprises oneor more properties that are identical to tinzaparin, including but notlimited to chemical structure, molecular weight, anticoagulant activity,and/or sulfation content properties. In another embodiment, theenzymatically-depolymerized LMW-HS product is substantially identical totinzaparin.

In another embodiment, the enzymatically-depolymerized LMW-HS productcan be formed from an N,2,3,6-HS product synthesized according to any ofthe methods of the present invention described above, according to thefollowing steps: (a) synthesizing an N,2,3,6-HS product according to anyof the above methods; (b) providing a reaction mixture comprising atleast one carbon-oxygen lyase; and (c) treating the N,2,3,6-HS productwith the carbon-oxygen lyase reaction mixture for a time sufficient todepolymerize at least a portion of the N,2,3,6-HS product, therebyforming the enzymatically-depolymerized LMW-HS product. In anotherembodiment, the enzymatically-depolymerized LMW-HS product comprises thestructure of Formula XI. In another embodiment, the N,2,3,6-HS productis an unfractionated N,2,3,6-HS product.

In another embodiment, the at least one carbon-oxygen lyase can be acarbon-oxygen lyase from any species, so long as the enzyme catalyzesβ-eliminative cleavage of HS polysaccharides. In another embodiment, theat least one carbon-oxygen lyase can be selected from the groupconsisting of the carbon-oxygen lyases from Bacteroides eggerthiicomprising the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 31, andSEQ ID NO: 32. In another embodiment, the at least one carbon-oxygenlyase can comprise one, two, or all three of the enzymes having theamino acid sequences of SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32,respectively.

In another embodiment, the time sufficient to form theenzymatically-depolymerized LMW-HS product is the time sufficient tocause the product to have a desired average molecular weight. In anotherembodiment, the M _(w) of the enzymatically-depolymerized LMW-HS productcan be in the range of 2,000 Da to 10,000 Da, preferably 5,500 Da to7,500 Da, and more preferably 6,500 Da. In another embodiment, theenzymatically-depolymerized LMW-HS product can have anticoagulantactivity. In another embodiment, the enzymatically-depolymerized LMW-HSproduct has an anti-Xa activity of at least 70 IU mg⁻¹ and up to 120 IUmg⁻¹, and/or a ratio of anti-Xa activity to anti-IIa activity of atleast 1.5:1, and up to 2.5:1.

In another embodiment, an N,2,3,6-HS product, synthesized according toany of the methods of the present invention, can be modified by achemical β-elimination reaction to form a chemically β-eliminative,LMW-HS product. Historically, chemically β-eliminative LMWH productshave been prepared by treating pharmaceutical heparin or its quaternaryammonium salt with a base. Under these conditions, chemicalβ-elimination takes place, forming the chemically β-eliminative LMW-HSproduct that contains a 4,5-unsaturated uronic acid residue at thenon-reducing end, a feature observed in enzymatically-depolymerized LWMHpolysaccharides comprising the structure of Formula XI (see Linhardt, R.J. and Gunay, N. S., above). Control of the reaction conditions has ledto the production of chemically β-eliminative LMWH compositions thathave either been approved for clinical use or been administered duringclinical trials are which are described in more detail below.

In a first non-limiting example, a chemically β-eliminative LMWHcomposition that has been prescribed for clinical use is bemiparin (CASNo: 91449-79-5; ATC code: B01AB12) (see e.g. Chapman, T. M. and Goa, K.L., (2003) Drugs 63 (21):2357-2377; Sanchez-Ferrer, C. F. (2010) Drugs70 Suppl. 2:19-23; Ciccone, M. M., et al., (2014) Vascular Pharmacology62:32-37). Bemiparin is prepared by alkaline depolymerization ofpharmaceutical heparin, particularly by reacting the benzethonium saltof pharmaceutical heparin with a quaternary ammonium hydroxide, such asTriton® B (benzyl trimethylammonium hydroxide), in the presence ofmethanol (see U.S. Pat. No. 4,981,955 and European Patent EP 0293539,the disclosures of which are incorporated by reference in theirentireties). Upon subsequent purification and precipitation, theresulting bemiparin composition comprising the structure of Formula XIhas an M _(w) in a range of at least 3,000 Da, up to 4,200 Da, andtypically 3,600 Da, and a size distribution such that: less than 35% ofthe polysaccharide chains have an M_(r) less than 2,000; a range of atleast 50% and up to 75% of the polysaccharide chains have an M_(r) in arange of at least 2,000 and up to 6,000; and less than 15% of thepolysaccharide chains have an M_(r) greater than 6,000. Additionally,bemiparin compositions can comprise an anti-Xa activity of at least 80IU mg⁻¹ and up to 120 IU mg⁻¹, an anti-IIa activity of at least 5 IUmg⁻¹ and up to 20 IU mg⁻¹, and/or a ratio of anti-Xa activity toanti-IIa activity of at least 8.0:1, and up to 10:1 (see Sanchez-Ferrer,C. F., above).

In another non-limiting example, a chemically β-eliminative LMWHcomposition that has been administered to patients during clinicaltrials is semuloparin (CAS No: 9041-08-1). Semuloparin is prepared byreacting the benzyl ester of a pharmaceutical heparin benzethonium saltwith the strong phosphazene base, BEMP(2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diaza-phosphorine),with subsequent saponification of the benzyl esters and purification(see Viskov, C., et al., (2009) J. Thromb. Haemost. 7:1143-1551).Phosphazene bases are among the strongest-known organic bases, by arehighly-sterically hindered and non-nucleophilic. As a result,phosphazene bases target the least sterically hindered regions of theheparin for β-elimination, and avoid the AT-recognition sequence thatcomprises the 3-O sulfated glucosamine residue. The resultingsemuloparin product having the structure of Formula XI has an M _(w) ina range of at least 2,000 Da, up to 3,000 Da, and typically 2,400 Da,and the anticoagulant activity of the semuloparin product comprises ananti-Xa activity of about 160 IU mg⁻¹, an anti-IIa activity of about 2IU mg⁻¹, and a ratio of anti-Xa activity to anti-IIa activity of about80:1 (see Viskov, C., above).

In another non-limiting example, a chemically β-eliminative LMWHcomposition that has been prescribed for clinical use is enoxaparin (CASNo: 679809-58-6; ATC code: B01AB05) (see e.g. Linhardt, R. J. and Gunay,N. S., above). Enoxaparin is prepared similarly to semuloparin in that abenzyl ester form of the pharmaceutical heparin is prepared, beforebeing reacted with a base. The benzyl ester is formed in a chlorinatedorganic solvent, such as chloroform or methylene chloride, in thepresence of a chlorine derivative, such as benzyl chloride, whichcontrols the amount of esterification in the resulting benzyl ester formof the pharmaceutical heparin (about 9-14% efficiency). Once the benzylester is formed, it is subsequently treated with a strong,non-sterically hindered base, such as sodium hydroxide, at hightemperature (see U.S. Pat. No. 5,389,618 and U.S. Reissue patentRE38,743, the disclosures of which are incorporated by reference intheir entireties. However, some (about 15% to 25%) polysaccharideswithin enoxaparin can additionally comprise a terminal 1,6-anhydro sugarresidue (either 1,6-anhydromannose or 1,6-anhydroglucosamine) at thereducing end, in addition to the characteristic 4,5-unsaturated uronicacid at the non-reducing end (see Guerrini, M., (2010) J. Med Chem.53:8030-8040). As a result, enoxaparin typically comprisespolysaccharides having the characteristic structure illustrated inFormula XII, below, in addition to polysaccharides comprising thestructure of Formula XI.

As illustrated above in Formula XII, n can be any integer from 1-21.Instead of a glucuronic acid or uronic acid residue, the sugar residueat the non-reducing end of enoxaparin polysaccharides is2-O-sulfo-4-enepyranosulfonic acid. Additionally, each glucosamineresidue at the reducing end comprises a 1,6-anhydro moiety, and thestereochemistry around the C2 carbon determines whether the residue is a1,6-anhydromannose or 1,6-anhydroglucosamine residue. Optionally, the3-O position of a glucosamine residue within one or more of disaccharideunits can also be 3-O sulfated. Without being limited by a particulartheory, it is believed that at least some of the polysaccharides withinenoxaparin comprises 3-O sulfated glucosamine residues, which ultimatelyleads to its anticoagulant activity.

As a commonly prescribed LMWH drug, compositions of enoxaparin that areadministered to patients must satisfy a series of stringent size,activity, and purity requirements established by both the EuropeanPharmacopoeia and the USP. (see “Enoxaparin Sodium” (2010) EuropeanPharmacopoeia 7.0, 1920-1921). In addition to comprising the structureof Formula XII above, properties that must be present in order tosatisfy the requirements include: an M _(w) in a range from at least3,800 Da, and up to 5,000 Da, and characteristically 4,500 Da; not lessthan 1.8 sulfate groups per disaccharide unit; and an anti-Xa activityof at least 90 IU mg⁻¹ and up to 125 IU mg⁻¹, an anti-IIa activity of atleast 20 IU mg⁻¹ and up to 35 IU mg⁻¹; and/or a ratio of anti-Xaactivity to anti-IIa activity of at least 3.3:1, and up to 5.3:1.Further, enoxaparin compositions suitable to be administered to patientscomprise a size distribution such that: at least 12.0%, up to 20.0%percent, and characteristically about 16%, of the polysaccharide chainshave an M_(r) less than 2,000; a range of at least 68.0%, up to 82.0%,and characteristically about 74%, of the polysaccharide chains have anM_(r) in a range of at least 2,000 and up to 8,000; and not more than18.0% of the polysaccharide chains have an M_(r) greater than 8,000.

Accordingly, in another embodiment, an N,2,3,6-HS product synthesizedaccording to any of the methods of the present invention described abovecan subsequently be depolymerized by one or more bases to form achemically β-eliminative LMW-HS product. In another embodiment, thechemically β-eliminative LMW-HS product comprises one or more propertiesthat are identical to bemiparin, including but not limited to chemicalstructure, molecular weight, anticoagulant activity, and/or sulfationcontent properties. In another embodiment, the chemically β-eliminativeLMW-HS product is substantially identical to bemiparin. In anotherembodiment, the chemically β-eliminative LMW-HS product comprises one ormore properties that are identical to semuloparin, including but notlimited to chemical structure, molecular weight, anticoagulant activity,and/or sulfation content properties. In another embodiment, thechemically β-eliminative LMW-HS product is substantially identical tosemuloparin. In another embodiment, the chemically β-eliminative LMW-HSproduct comprises one or more properties that are identical toenoxaparin, including but not limited to chemical structure, molecularweight, anticoagulant activity, and/or sulfation content properties. Inanother embodiment, the chemically β-eliminative LMW-HS product issubstantially identical to enoxaparin.

In another embodiment, the chemically β-eliminative LMW-HS product canbe formed from an N,2,3,6-HS product synthesized according to any of themethods of the present invention described above, according to thefollowing steps: (a) synthesizing an N,2,3,6-HS product according to anyof the above methods; (b) providing a reaction mixture comprising abase; and (c) treating the N,2,3,6-HS product with the reaction mixturecomprising the base for a time sufficient to depolymerize at least aportion of the N,2,3,6-HS product, thereby forming the chemicallyβ-eliminative LMW-HS product. In another embodiment, the chemicallyβ-eliminative LMW-HS product comprises the structure of Formula XI. Inanother embodiment, the chemically β-eliminative LMW-HS productcomprises the structure of Formula XII. In another embodiment, theN,2,3,6-HS product is an unfractionated N,2,3,6-HS product.

In another embodiment, the base is Triton® B, and the step of treatingthe N,2,3,6-HS product with the reaction mixture comprising Triton® Bfurther comprises the following sub-steps: (i) reacting theunfractionated N,2,3,6-HS product with a benzethonium salt, preferablybenzethonium chloride, to form a benzethonium HS salt; and (ii)combining the benzethonium HS salt with a reaction mixture comprisingTriton® B and methanol, to form the chemically β-eliminative LMW-HSproduct. In another embodiment, the sub-step of preparing the chemicallyβ-eliminative LMW-HS product from the benzethonium HS salt comprises theprocedure reported in any of the examples in U.S. Pat. No. 4,981,955,preferably Example 3. In another embodiment, the time sufficient todepolymerize the benzethonium HS salt is the time sufficient to form achemically β-eliminative LMW-HS product to having an M _(w) in a rangeof at least 3,000 Da, up to 4,200 Da, and preferably 3,600 Da, andhaving a size distribution such that: less than 35% of thepolysaccharide chains have an M_(r) less than 2,000; a range of at least50% and up to 75% of the polysaccharide chains have an M_(r) in a rangeof at least 2,000 and up to 6,000; and less than 15% of thepolysaccharide chains have an M_(r) greater than 6,000. In anotherembodiment, the chemically β-eliminative LMW-HS product comprises thestructure of Formula XI. In another embodiment, the chemicallyβ-eliminative LMW-HS product comprises an anti-Xa activity of at least80 IU mg⁻¹ and up to 120 IU mg⁻¹, an anti-IIa activity of at least 5 IUmg⁻¹ and up to 20 IU mg⁻¹, and/or a ratio of anti-Xa activity toanti-IIa activity of at least 8.0:1, and up to 10:1. In anotherembodiment, the chemically β-eliminative LMW-HS product is substantiallyequivalent to bemiparin.

In another embodiment, the base is BEMP, and the step of treating theN,2,3,6-HS product with the reaction mixture comprising BEMP furthercomprises the following steps: (i) reacting the unfractionatedN,2,3,6-HS product with a benzethonium salt, preferably benzethoniumchloride, to form a benzethonium HS salt; (ii) esterification of thebenzethonium HS salt using benzyl chloride to form a benzyl ester HS;(iii) transalification of the benzyl ester HS with a benzethonium salt,preferably benzethonium chloride, to form a benzethonium benzyl esterHS; (iv) depolymerization of the benzethonium benzyl ester HS with BEMPto form a benzyl ester chemically β-eliminative LMW-HS product; and (v)saponification of the benzyl ester chemically β-eliminative LMW-HSproduct to form the chemically β-eliminative LMW-HS product, as reportedin Viskov, C., et al., above. In another embodiment, the time sufficientto depolymerize the benzethonium benzyl ester HS with BEMP is the timesufficient to form a benzyl ester chemically β-eliminative LMW-HSproduct such that upon saponification of the benzyl esters, theresulting chemically β-eliminative LMW-HS product has an M _(w) in arange of at least 2,000 Da, up to 3,000 Da, and preferably about 2,400Da. In another embodiment, the chemically β-eliminative LMW-HS productcomprises the structure of Formula XI. In another embodiment, thechemically β-eliminative LMW-HS product comprises an anti-Xa activity ofabout 160 IU mg⁻¹, an anti-IIa activity of about 2 IU mg⁻¹, and/or aratio of anti-Xa activity to anti-IIa activity of at least 80:1, and upto 100:1. In another embodiment, the chemically β-eliminative LMW-HSproduct is substantially equivalent to semuloparin.

In another embodiment, the base is sodium hydroxide, and the step oftreating the N,2,3,6-HS product with the reaction mixture comprisingsodium hydroxide further comprises the following sub-steps: (i) reactingthe unfractionated N,2,3,6-HS product with a benzethonium salt,preferably benzethonium chloride, to form a benzethonium HS salt; (ii)esterification of the benzethonium HS salt using benzyl chloride in thepresence of a chlorinated solvent, preferably methylene chloride orchloroform, to form a benzyl ester HS; and (iii) combining the benzylester HS with a reaction mixture comprising sodium hydroxide to form thechemically β-eliminative LMW-HS product. In another embodiment, thebenzyl ester HS has a degree of esterification of at least 9%, and up toabout 14%. In another embodiment, the reaction between the benzyl esterHS and sodium hydroxide is performed at a temperature selected withinthe range of at least 50° C., up to 70° C., and preferably within therange of at least 55° C., and up to 65° C. In another embodiment, thebenzyl ester HS and chemically β-eliminative LMW-HS product are preparedaccording to the procedure of Example 3 within U.S. RE38,743. In anotherembodiment, the time sufficient to depolymerize the benzyl ester HS isthe time sufficient to form a chemically β-eliminative LMW-HS product tohaving an M _(w) in a range of at least 3,800 Da, up to 5,000 Da, andpreferably 4,500 Da. In another embodiment, the chemically β-eliminativeLMW-HS product comprises a size distribution such that: at least 12.0%,up to 20.0% percent, and preferably about 16%, of the polysaccharidechains have an M_(r) less than 2,000; a range of at least 68.0%, up to82.0%, and preferably about 74%, of the polysaccharide chains have anM_(r) in a range of at least 2,000 and up to 8,000; and not more than18.0% of the polysaccharide chains have an M_(r) greater than 8,000. Inanother embodiment, the chemically β-eliminative LMW-HS productcomprises not less than 1.8 sulfate groups per disaccharide unit. Inanother embodiment, the chemically β-eliminative LMW-HS productcomprises an anti-Xa activity of at least 90 IU mg⁻¹ and up to 125 IUmg⁻¹, an anti-IIa activity of at least 20 IU mg⁻¹ and up to 35 IU mg⁻¹;and/or a ratio of anti-Xa activity to anti-IIa activity of at least3.3:1, and up to 5.3:1. In another embodiment, the chemicallyβ-eliminative LMW-HS product is substantially equivalent to enoxaparin.

In another embodiment, an N,2,3,6-HS product, synthesized according toany of the methods of the present invention, can be modified by adeamination reaction to form a deaminated LMW-HS product. Historically,deaminated LMWH products have been prepared by treating pharmaceuticalheparin with nitrous acid. Under these conditions, a deaminated LMWHproduct is formed that contains a 2-O-sulfo-α-L-idopyranosuronic acidresidue at the non-reducing end and a 6-O-sulfo-2,5-anhydro-D-mannitolresidue at the reducing end (see Linhardt, R. J. and Gunay, N. S.,above). Deaminated LMWH products comprising2-O-sulfo-α-L-idopyranosuronic acid residues at the non-reducing end and6-O-sulfo-2,5-anhydro-D-mannitol residues at the reducing end generallycomprise the structure of Formula XIII, below:

As illustrated above in Formula XIII, n can be any integer from 3-20,and Y can be an aldehyde, hydroxyl, or carboxylic acid functional group.In another embodiment, Y is a hydroxyl group. Optionally, the 3-Oposition of a glucosamine residue within one or more of disaccharideunits can also be 3-O sulfated. Without being limited by a particulartheory, it is believed that at least some of the polysaccharides withinthe deaminated LMWH product comprises 3-O sulfated glucosamine residues,which ultimately leads to its anticoagulant activity.

Non-limiting examples of deaminated LMWH compositions that have beenprescribed for clinical use include dalteparin (CAS No: 9041-08-1; ATCcode: B01AB04), nadroparin (CAS No: 9005-49-6; ATC code: B01AB06),reviparin (CAS No: 9005-49-6; ATC code: B01AB08) and certoparin (CAS No:9005-49-6). Generally, each of dalteparin, nadroparin, and reviparin areprepared by depolymerization using nitrous acid, either added directlyor formed in situ by the addition of sodium nitrite to an acidiccomposition. Certoparin is prepared similarly, using a nitrous acidderivative such as isoamyl nitrite (see Linhardt, R. J. and Gunay, N.S., above). Control of the reaction conditions has led to the productionof deaminated LMW-HS compositions that have slightly differentanticoagulant activities and molecular weight properties relative toeach other, and described, for example, in U.S. Pat. Nos. 4,303,651,4,351,938, 4,438,261, 4,500,519, 4,686,388, 5,019,649, and 5,599,801,the disclosures of which are incorporated by reference in theirentireties.

In a first non-limiting example, a deaminated LMWH composition that hasbeen prescribed for clinical use is dalteparin (see e.g. Jacobsen, A.F., et al., (2003) Br J Obstet Gynaecol 110:139-144; and Guerrini, M.,et al., (2007) Seminars in Thrombosis and Hemostasis 33 (5):478-487).Dalteparin is typically prepared as a sodium salt by an aciddepolymerization of pharmaceutical heparin, particularly by reactingpharmaceutical heparin with nitrous acid (see e.g. U.S. Pat. No.5,019,649). Upon subsequent purification and precipitation, theresulting dalteparin composition comprising the structure of FormulaXIII has an M _(w) in a range of at least 5,600 Da, up to 6,400 Da, andtypically 6,000 Da, and a size distribution such that the proportion ofpolysaccharide chains having an M_(r) less than 3,000 is not more than13.0%; and at least 15.0% and up to 25.0% of the chains have an M_(r) ofat least 8,000. Additionally, dalteparin compositions can comprise ananti-Xa activity of at least 110 IU mg⁻¹ and not more than 210 IU mg⁻¹,an anti-IIa activity of at least 35 IU mg⁻¹ and not more than 100 IUmg⁻¹, and/or a ratio of anti-Xa activity to anti-IIa activity of atleast 1.9:1, and up to 3.2:1 (see “Dalteparin Sodium” (2010) EuropeanPharmacopoeia 7.0, 1788-1789).

In another non-limiting example, a deaminated LMWH composition that hasbeen prescribed for clinical use is nadroparin. Nadroparin is commonlyprepared as a sodium or calcium salt by an acid depolymerization ofpharmaceutical heparin, using sodium nitrite in the presence ofhydrochloric acid to maintain a pH of about 2.5 (see e.g. U.S. Pat. Nos.4,686,388 and 5,599,801) the disclosures of which are incorporated byreference in their entireties). Upon subsequent purification andprecipitation, the resulting nadroparin composition comprising thestructure of Formula XIII has an M _(w) in a range of at least 3,600 Da,up to 5,000 Da, and typically 4,300 Da, and a size distribution suchthat the proportion of chains having an M_(r) less than 2,000 is notmore than 15%; and at least 75% and up to 95% of the chains have anM_(r) in a range of at least 2,000 and up to 8,000, with at least 35%and up to 55% of the chains having an M_(r) of at least 2,000 and up to4,000. Additionally, nadroparin compositions can comprise an anti-Xaactivity of not less than 95 IU mg⁻¹ and not more than 130 IU mg⁻¹,and/or a ratio of anti-Xa activity to anti-IIa activity of at least2.5:1, and up to 4.0:1 (see “Nadroparin Sodium” (2010) EuropeanPharmacopoeia 7.0, 1788-1789).

Other non-limiting examples of deaminated LMWH compositions that havebeen prescribed for clinical use is reviparin and certoparin. Reviparinis prepared similarly to dalteparin and nadroparin, by introducingnitrous acid or forming nitrous acid in situ (see Linhardt, R. J. andGunay, N. S., above), and the resulting reviparin composition comprisingthe structure of Formula XIII has an M _(w) in a range of at least 4,200Da, up to 4,600 Da, and typically 4,400 Da, and a ratio of anti-Xaactivity to anti-IIa activity of at least 4.0:1, up to 4.5:1, andtypically 4.2:1 (see Grey, et al, above). Certoparin is prepared byreacting heparin with isoamyl nitrite in the presence of acetic orhydrochloric acid (see Ahsan, A., et al., (2000) Clin. Appl.Thrombosis/Hemostasis 6 (3): 169-174). The resulting certoparincomposition comprising the structure of Formula XIII has an M _(w) in arange of at least 5,000 Da, up to 5,600 Da, and typically 5,400 Da, anda ratio of anti-Xa activity to anti-IIa activity of at least 2.0:1, upto 2.5:1, and preferably 2.4:1 (see Grey, et al, above).

Accordingly, in another embodiment, an N,2,3,6-HS product synthesizedaccording to any of the methods of the present invention described abovecan subsequently be depolymerized by nitrous acid, or a nitrous acidderivative such as isoamyl nitrite, to form a deaminated LMW-HS product.In another embodiment, the deaminated LMW-HS product comprises one ormore properties that are identical to dalteparin, including but notlimited to chemical structure, molecular weight, anticoagulant activity,and/or sulfation content properties. In another embodiment, thedeaminated LMW-HS product is substantially identical to dalteparin. Inanother embodiment, the deaminated LMW-HS product comprises one or moreproperties that are identical to nadroparin, including but not limitedto chemical structure, molecular weight, anticoagulant activity, and/orsulfation content properties. In another embodiment, the deaminatedLMW-HS product is substantially identical to nadroparin. In anotherembodiment, the deaminated LMW-HS product comprises one or moreproperties that are identical to reviparin, including but not limited tochemical structure, molecular weight, anticoagulant activity, and/orsulfation content properties. In another embodiment, the deaminatedLMW-HS product is substantially identical to reviparin. In anotherembodiment, the deaminated LMW-HS product comprises one or moreproperties that are identical to certoparin, including but not limitedto chemical structure, molecular weight, anticoagulant activity, and/orsulfation content properties. In another embodiment, the deaminatedLMW-HS product is substantially identical to certoparin.

In another embodiment, the deaminated LMW-HS product can be formed froman N,2,3,6-HS product synthesized according to any of the methods of thepresent invention described above, according to the following steps: (a)synthesizing an N,2,3,6-HS product according to any of the abovemethods; (b) providing a deamination reaction mixture comprising adeamination agent, preferably a deamination agent selected from thegroup consisting of isoamyl nitrate and nitrous acid; and (c) treatingthe N,2,3,6-HS product with the deamination reaction mixture for a timesufficient to depolymerize at least a portion of the N,2,3,6-HS product,thereby forming the deaminated LMW-HS product. In another embodiment,the deamination agent is nitrous acid, the deamination reaction mixturecan comprise stoichiometric quantities of an acid, preferably aceticacid or hydrochloric acid, and an alkali or alkaline earth metal nitritesalt, preferably sodium nitrite, wherein the nitrous acid is formedwithin the deamination reaction mixture in situ. In another embodiment,the deamination agent is isoamyl nitrite. In another embodiment, thedeaminated LMW-HS product comprises the structure of Formula XIII Inanother embodiment, the N,2,3,6-HS product is an unfractionatedN,2,3,6-HS product.

In another embodiment, the time sufficient to form the deaminated LMW-HSproduct is the time sufficient to cause the product to have a desiredaverage molecular weight. In another embodiment, the M _(w) of thedeaminated LMW-HS product is in the range of 2,000 Da to 10,000 Da,preferably in the range of 4,000 Da to 6,000 Da. In another embodiment,the M _(w) of the deaminated LMW-HS product is in the range 4,000 Da to4,500 Da, preferably 4,300 Da. In another embodiment, the M _(w) of thedeaminated LMW-HS product is in the range 4,200 Da to 4,600 Da,preferably 4,400 Da. In another embodiment, the M _(w) of the deaminatedLMW-HS product is in the range 5,000 Da to 5,600 Da, preferably 5,400Da. In another embodiment, the M _(w) of the deaminated LMW-HS productis in the range 5,700 Da to 6,300 Da, preferably 6,000 Da.

In another embodiment, the deaminated LMW-HS product can haveanticoagulant activity. In another embodiment, the deaminated LMW-HSproduct has an anti-Xa activity of up to 210 IU mg⁻¹. In anotherembodiment, the deaminated LMW-HS product has an anti-Xa activity of atleast 110 IU mg⁻¹ and not more than 210 IU mg⁻¹. In another embodiment,the deaminated LMW-HS product has an anti-Xa activity of not less than95 IU mg⁻¹ and not more than 130 IU mg⁻¹. In another embodiment, thedeaminated LMW-HS product has an anti-IIa activity of at least 35 IUmg⁻¹ and not more than 100 IU mg⁻¹. In another embodiment, thedeaminated LMW-HS product has a ratio of anti-Xa activity to anti-IIaactivity of at least 2.0:1, and up to 4.5:1. In another embodiment, thedeaminated LMW-HS product has a ratio of anti-Xa activity to anti-IIaactivity of at least 3.0:1, and up to 3.6:1. In another embodiment, thedeaminated LMW-HS product has a ratio of anti-Xa activity to anti-IIaactivity of at least 4.0:1, and up to 4.5:1. In another embodiment, thedeaminated LMW-HS product has a ratio of anti-Xa activity to anti-IIaactivity of at least 2.0:1, and up to 2.5:1. In another embodiment, thedeaminated LMW-HS product has a ratio of anti-Xa activity to anti-IIaactivity of at least 2.2:1, and up to 2.7:1.

In another embodiment, an N,2,3,6-HS product, synthesized according toany of the methods of the present invention, can be modified by anoxidation reaction to form an oxidized LMW-HS product. Historically,oxidized LMWH products have been prepared by treating pharmaceuticalheparin with an acid, and then reacting the acidified heparin with anoxidizing agent, particularly a peroxide or a superoxide compound suchas hydrogen peroxide, at an elevated temperature. Under theseconditions, an oxidized LMWH product can be formed that retains thestructure of pharmaceutical heparin, particularly comprising thestructure of Formula I, but is in the same approximate molecular weightand anticoagulant activity ranges as other LMW-HS compounds.

Control of the reaction conditions has led to the production of oxidizedLMWH compositions that have different anticoagulant activities andmolecular weight properties relative to each other, and described, forexample, in U.S. Pat. Nos. 4,281,108, 4,629,699, and 4,791,195, as wellas European Patent EP0101141, the disclosures of which are incorporatedby reference in their entireties. In particular, the acidified heparinhas been formed by reacting the pharmaceutical heparin with a strongacid, such as hydrochloric acid, or a weak acid, such as ascorbic acid.Acidified heparin has also been formed by binding pharmaceutical heparinto a strong cationic exchange resin. Similarly, the depolymerizationconditions can be controlled with respect to the pH and temperature atwhich the depolymerization takes place, and the oxidizing agent itself.

Non-limiting examples of oxidized LMWH compositions that have beenprescribed for clinical use include parnaparin (CAS No: 91449-79-5; ATCcode: B01AB05) and ardeparin (CAS No: 9005-49-6). In particular,Parnaparin has been used in the prevention of venous thromboembolism, inthe treatment of chronic venous disorders, and in the treatment ofvenous and arterial thrombosis (see e.g. Camporese, G., et al., (2009)Vascular Health and Risk Management 5:819-831). Without being limited bya particular theory, it is believed that parnaparin is produced byforming the acidified heparin using ascorbic acid, and subsequentlydepolymerizing the acidified heparin under slightly basic conditions inthe presence of cupric acetate monohydrate and hydrogen peroxide withincubation at 50° C. (see U.S. Pat. No. 4,791,195, Example 1).Parnaparin that has been administered to patients has an M _(w) in arange of at least 4,000 Da, up to 6,000 Da, and typically 5,000 Da, anda size distribution such that the proportion of polysaccharides havingan M_(r) less than 3,000 is not more than 30% of the composition, andthe proportion of polysaccharides having an M_(r) in a range of at least3,000 and up to 8,000 is between 50% and 60% of the composition.Additionally, parnaparin compositions can comprise an anti-Xa activityof at least 75 IU mg⁻¹ and not more than 110 IU mg⁻¹, and/or a ratio ofanti-Xa activity to anti-IIa activity of at least 1.5:1, and up to 3.0:1(see “Parnaparin Sodium” (2010) European Pharmacopoeia 7.0, 2672). Onthe other hand, ardeparin compositions that have been prescribed topatients have generally had an M _(w) in a range of at least 5,500 Da,up to 6,500 Da, and typically 6,000 Da, an anti-Xa activity of 120+/−25IU mg⁻¹, and a ratio of anti-Xa activity to anti-IIa activity of atleast 2.0:1, up to 2.5:1, and characteristically 2.3:1.

Accordingly, in another embodiment, an N,2,3,6-HS product synthesizedaccording to any of the methods of the present invention described abovecan subsequently be depolymerized by an oxidizing agent to form anoxidized LMW-HS product. In another embodiment, the oxidized LMW-HSproduct comprises one or more properties that are identical toparnaparin, including but not limited to chemical structure, molecularweight, anticoagulant activity, and/or sulfation content properties. Inanother embodiment, the oxidized LMW-HS product is substantiallyidentical to parnaparin. In another embodiment, the oxidized LMW-HSproduct comprises one or more properties that are identical toardeparin, including but not limited to chemical structure, molecularweight, anticoagulant activity, and/or sulfation content properties. Inanother embodiment, the oxidized LMW-HS product is substantiallyidentical to ardeparin.

In another embodiment, the oxidized LMW-HS product can be formed from anN,2,3,6-HS product synthesized according to any of the methods of thepresent invention described above, according to the following steps: (a)synthesizing an N,2,3,6-HS product according to any of the abovemethods; (b) providing an oxidation reaction mixture comprising anoxidation agent, preferably hydrogen peroxide; and (c) treating theN,2,3,6-HS product with the oxidation reaction mixture for a timesufficient to depolymerize at least a portion of the N,2,3,6-HS product,thereby forming the oxidized LMW-HS product. In another embodiment, thestep of treating the N,2,3,6-HS product with the oxidation reactionmixture can comprise the following sub-steps: (i) acidifying theN,2,3,6-HS product to form an acidified N,2,3,6-HS product; (ii)combining the acidified HS product with the oxidation reaction mixture;and (c) incubating the acidified HS product within the oxidationreaction mixture at a temperature of at least 50° C. until an oxidizedLMW-HS product is formed. In another embodiment, the step of treatingthe N,2,3,6-HS product with the oxidation reaction mixture can comprisethe procedure of Example 1 of U.S. Pat. No. 4,791,195. In anotherembodiment, the oxidized LMW-HS product comprises the structure ofFormula I. In another embodiment, the N,2,3,6-HS product is anunfractionated N,2,3,6-HS product.

In another embodiment, the time sufficient to form the oxidized LMW-HSproduct is the time sufficient to cause the product to have a desiredaverage molecular weight. In another embodiment, the M _(w) of theoxidized LMW-HS product is in the range of 2,000 Da to 12,000 Da,preferably in the range of 4,000 Da to 6,500 Da. In another embodiment,the M _(w) of the oxidized LMW-HS product is in the range 4,000 Da to6,000 Da, preferably 5,000 Da. In a further embodiment, the oxidizedLMW-HS product comprises a size distribution such that the proportion ofpolysaccharides having an M_(r) less than 3,000 is not more than 30% ofthe composition, and the proportion of polysaccharides having an M_(r)in a range of at least 3,000 and up to 8,000 is between 50% and 60% ofthe composition. In another embodiment, the M _(w) of the oxidizedLMW-HS product is in the range 5,500 Da to 6,500 Da, preferably 6,000Da.

In another embodiment, the oxidized LMW-HS product can haveanticoagulant activity. In another embodiment, the oxidized LMW-HSproduct has an anti-Xa activity of at least 75 IU mg⁻¹. In anotherembodiment, the oxidized LMW-HS product has an anti-Xa activity of notmore than 110 IU mg⁻¹. In another embodiment, the oxidized LMW-HSproduct has a ratio of anti-Xa activity to anti-IIa activity of at least1.5:1, and up to 3.0:1. In another embodiment, the oxidized LMW-HSproduct has a ratio of anti-Xa activity to anti-IIa activity of at least2.0:1, up to 2.5:1, and preferably 2.3:1.

Those skilled in the art would appreciate that the examples describedabove of LMW-HS compositions, and methods for forming them from anN,2,3,6-HS product synthesized using one or more engineered arylsulfate-dependent sulfotransferase enzymes, are non-exhaustive, and thatsuch other examples are excluded for clarity and brevity. Once anN,2,3,6-HS product, particularly an unfractionated N,2,3,6-HS product,is formed according to any of the methods described above, it can bemodified and/or depolymerized by any known process to form a secondaryproduct, particularly an LMW-HS product. Such processes include, but arenot limited to: fractionation using solvents (French Patent No.2,440,376, U.S. Pat. No. 4,692,435); fractionation using an anionicresin (French Patent No. 2,453,875); gel filtration; affinitychromatography (U.S. Pat. No. 4,401,758); controlled depolymerization bymeans of a chemical agent including, but not limited to, nitrous acid(European Patent EP 0014184, European Patent EP 0037319, European PatentEP 0076279, European Patent EP 0623629, French Patent No. 2,503,714,U.S. Pat. No. 4,804,652 and PCT Publication No. WO 81/03276),β-elimination from a heparin ester (European Patent EP 0040144, U.S.Pat. No. 5,389,618), periodate (European Patent EP 0287477), sodiumborohydride (European Patent EP 0347588, European Patent EP 0380943),ascorbic acid (U.S. Pat. No. 4,533,549), hydrogen peroxide (U.S. Pat.Nos. 4,629,699, 4,791,195), quaternary ammonium hydroxide from aquaternary ammonium salt of heparin (U.S. Pat. No. 4,981,955), alkalimetal hydroxide (European Patent EP 0380943, European Patent EP0347588), using carbon-oxygen lyase enzymes (European Patent EP 0064452,U.S. Pat. No. 4,396,762, European Patent EP 0244235, European Patent EP0244236; U.S. Pat. Nos. 4,826,827; 3,766,167), by means of irradiation(European Patent EP 0269981), purification and modification offast-moving HS fractions (U.S. Pat. Nos. 7,687,479, 8,609,632), andother methods or combinations of methods such as those described in U.S.Pat. Nos. 4,303,651, 4,757,057, U.S. Publication No. 2007/287683, PCTPublication No. WO 2009/059284 and PCT Publication No. WO 2009/059283,the disclosures of which are incorporated by reference in theirentireties.

Preparation of Engineered Aryl Sulfate Dependent SulfotransferaseEnzymes

In general, the engineered sulfotransferases encoded by the disclosednucleic acid and amino acid sequences can be expressed and purifiedusing any microbiological technique known in the art, including asdescribed below. The aryl sulfate-dependent sulfotransferase activity ofeach purified enzyme can be determined spectrophotometrically orfluorescently and/or using mass spectrometry (MS) or nuclear magneticresonance (NMR) spectroscopy to characterize the starting materialsand/or sulfated polysaccharide products. Such methods are describedbelow in the Examples section.

The engineered gene products, proteins and polypeptides utilized inaccordance with methods of the present invention can also includeanalogs that contain insertions, deletions, or mutations relative to thedisclosed DNA or peptide sequences, and that also encode for enzymesthat catalyze reactions in which aryl sulfate compounds are substrates.In another embodiment, each analog similarly catalyzes sulfotransferreactions in which aryl sulfate compounds are utilized as sulfo donors.Analogs can be derived from nucleotide or amino acid sequences asdisclosed herein, or they can be designed synthetically in silico or denovo using computer modeling techniques. Those skilled in the art willappreciate that other analogs, as yet undisclosed or undiscovered, canbe used to design and/or construct different sulfate-dependentsulfotransferase enzymes capable of being utilized in accordance withmethods of the present invention. There is no need for a gene product,protein, or polypeptide to comprise all or substantially all of anucleic acid or amino acid sequence of an engineered sulfotransferase asdisclosed herein. Such sequences are herein referred to as “segments.”Further, the gene products, proteins, and polypeptides discussed anddisclosed herein can also include fusion or recombinant arylsulfate-dependent sulfotransferases comprising full-length sequences orbiologically functional segments of sequences disclosed in the presentinvention. Methods of preparing such proteins are known in the art.

In addition to the nucleic acid and amino acid sequences disclosedherein, methods of the present invention can be practiced by arylsulfate-dependent sulfotransferases comprising amino acid sequences thatare substantially identical to any of the disclosed amino acid sequencesabove, or expressed from nucleic acids comprising a nucleotide sequencethat is substantially identical to a disclosed nucleotide sequence (SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27). Thoseskilled in the art can determine appropriate nucleotide sequences thatencode for polypeptides having the amino acid sequence of SEQ ID NOs:33-54 and 56-61, based on the nucleotide sequences above. “Substantiallyidentical” sequences, as used in the art, refer to sequences whichdiffer from a particular reference sequence by one or more deletions,substitutions, or additions, the net effect of which is to retain atleast some of the biological activity of the engineered polypeptideencoded by the reference sequence. Namely, the biological activity ofthe engineered aryl sulfate-dependent sulfotransferases comprises thetransfer of a sulfo group from a sulfo donor aryl sulfate compound to apolysaccharide acting as a sulfo group acceptor. In another embodiment,the polysaccharide is a heparosan-based and/or HS polysaccharide.Accordingly, as used to describe the aryl sulfate-dependent enzymes ofthe present invention, “substantial identity” can refer either toidentity with a particular gene product, polypeptide or amino acidsequence of an aryl sulfate-dependent enzyme, or a gene or nucleic acidsequence encoding for an aryl sulfate-dependent enzyme. Such sequencescan include mutations of the disclosed sequences or a sequence in whichthe biological activity is altered, enhanced, or diminished to somedegree but retains at least some of the original biological activity ofa disclosed reference amino acid sequence or polypeptide encoded by adisclosed reference nucleic acid sequence.

Alternatively, DNA analog sequences are substantially identical to thespecific DNA sequences disclosed herein if: (a) the DNA analog sequenceis derived from coding regions of the any of the disclosed nucleic acidsequences; or (b) the DNA analog sequence is capable of hybridization ofDNA sequences of (a) under stringent conditions and which encodebiologically active aryl sulfate-dependent sulfotransferase geneproduct; or (c) the DNA sequences are degenerate as a result ofalternative genetic code to the DNA analog sequences defined in (a)and/or (b). Substantially identical analog proteins will be greater thanabout 60% identical to the corresponding sequence of the native protein.Sequences having lesser degrees of identity but comparable biologicalactivity, namely, transferring a sulfo group from an aryl sulfatecompound to polysaccharides, particularly heparosan-based or HSpolysaccharides, are also considered to be substantially identical. Indetermining the substantial identity of nucleic acid sequences, allsubject nucleic acid sequences capable of encoding substantiallyidentical amino acid sequences are considered to be substantiallyidentical to a reference nucleic acid sequence, regardless ofdifferences in codon sequences or amino acid substitutions to createbiologically functional equivalents.

At a biological level, identity is just that, i.e. the same amino acidat the same relative position in a given family member of a gene family.Homology and similarity are generally viewed as broader terms. Forexample, biochemically similar amino acids, for example leucine andisoleucine or glutamic acid/aspartic acid, can be alternatively presentat the same position—these are not identical per se, but arebiochemically “similar.” As disclosed herein, these are referred to asconservative differences or conservative substitutions. This differsfrom a conservative mutation at the DNA level, which changes thenucleotide sequence without making a change in the encoded amino acid,e.g., TCC to TCA, both of which encode serine.

In some embodiments, the genes and gene products include within theirrespective sequences a sequence “essentially as that” of a gene encodingfor an aryl sulfate-dependent sulfotransferase or its correspondingprotein. A sequence essentially as that of a gene encoding for an arylsulfate-dependent sulfotransferase refers to sequences that aresubstantially identical or substantially similar to a portion of adisclosed nucleic acid sequence and contains a minority of bases oramino acids (whether DNA or protein) that are not identical to those ofa disclosed protein or a gene, or which are not a biologicallyfunctional equivalent. Biological functional equivalence is wellunderstood in the art and is further discussed in detail below.Nucleotide sequences are “essentially the same” where they have betweenabout 75% and about 85%, or particularly, between about 86% and about90%, or more particularly greater than 90%, or even more particularlybetween about 91% and about 95%, or still more particularly, betweenabout 96% and about 99%, of nucleic acid residues which are identical tothe nucleotide sequence of a disclosed gene. Similarly, peptidesequences which have about 80%, or 90%, or particularly from 90-95%, ormore particularly greater than 96%, or even more particularly 95-98%, orstill more particularly 99% or greater amino acids which are identicalor functionally equivalent or biologically functionally equivalent tothe amino acids of a disclosed polypeptide sequence will be sequenceswhich are “essentially the same.”

Additionally, alternate nucleic acid sequences that include functionallyequivalent codons are also encompassed by this invention. Functionallyequivalent codons refer to codons that encode the same amino acid, suchas the ACG and AGU codons for serine. Thus, substitution of afunctionally equivalent codon into any of the nucleotide sequences aboveencode for biologically functionally equivalent sulfotransferases. Thus,the present invention includes amino acid and nucleic acid sequencescomprising such substitutions but which are not set forth herein intheir entirety for convenience.

Those skilled in the art would recognize that amino acid and nucleicacid sequences can include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ nucleic acid sequences, and yet stillbe essentially as set forth in one of the sequences disclosed herein, solong as the sequence retains its biological activity with respect tobinding and reacting with aryl sulfate compounds as sulfo donors. Theaddition of terminal sequences particularly applies to nucleic acidsequences which can, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or caninclude various internal sequences, or introns, which are known to occurwithin genes.

As discussed above, modifications and changes can be made in thesequence of any of the disclosed aryl sulfate-dependentsulfotransferases, including conservative and non-conserved mutations,deletions, and additions while still constituting a molecule having likeor otherwise desirable characteristics. For example, certain amino acidscan be substituted for other amino acids in a protein structure withoutappreciable loss of interactive capacity with particular structures orcompounds, particularly aryl sulfate compounds and/or sulfo acceptorpolysaccharides. This can occur because the ability of a protein torecognize, bind, and react with other structures or compounds within itsenvironment defines that protein's biological functional activity, notthe sequence itself. Consequently, certain amino acid sequencesubstitutions can be made in that protein's sequence to obtain a proteinwith the equal, enhanced, or diminished properties. One non-limitingexample of such amino acid substitutions that can occur without anappreciable loss of interactive activity include substitutions inexternal domains or surfaces of the protein that do not affect thefolding and solubility of the protein. Similarly, amino acids canpotentially be added to either terminus of the protein so long as theability of the protein to fold or to recognize and bind its substratesis not deleteriously affected. One skilled in the art can appreciatethat several other methods and/or strategies can be utilized to alter anenzyme's sequence without affecting its activity.

Consequently, mutations, deletions, additions, or other alterations to aparent enzyme's structure or sequence in which the modified enzymeretains the parent enzyme's biological activity can be defined to bebiologically functionally equivalent to the parent enzyme. Thus,biologically functional equivalent enzymes, with respect to theengineered aryl sulfate-dependent sulfotransferases, can include anysubstitution or modification of any of the amino acid sequencesdisclosed herein, so long as the resultant modified enzyme is dependenton interacting with aryl sulfate compounds, particularly PNS or NCS, tocatalyze sulfo transfer to polysaccharides, particularly heparosan-basedand/or HS polysaccharides. In particular, such substitutions ormodifications can result from conservative mutations in the amino acidsequence in any portion of the protein, as described below, althoughnon-conservative mutations in non-catalytically active regions of theenzyme are also contemplated. Consequently, engineered arylsulfate-dependent sulfotransferases suitable to practice the methods ofthe present invention can be expressed from any nucleic acid having anucleotide sequence that encodes for a biologically functionalequivalent enzyme, although such nucleotide sequences are not set forthherein in their entirety for convenience.

Alternatively, recombinant DNA technology can be used to createbiologically functionally equivalent proteins or peptides in whichchanges in the protein structure can be engineered, based onconsiderations of the properties of the amino acids being exchanged.Rationally-designed changes can be introduced through the application ofsite-directed mutagenesis techniques, for example, to test whethercertain mutations affect positively or negatively affect the enzyme'saryl sulfate-dependent catalytic activity or binding of sulfo donors oracceptors within the enzyme's active site.

Amino acid substitutions, such as those which might be employed inmodifying any of the aryl sulfate-dependent sulfotransferases describedherein, are generally based on the relative similarity of the amino acidside-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, and the like. Those skilled in the art arefamiliar with the similarities between certain amino acids, such as thesize, shape and type of the amino acid side-chain substituents.Non-limiting examples include relationships such as that arginine,lysine and histidine are all positively charged residues; that alanine,glycine and serine are all of similar size; and that phenylalanine,tryptophan and tyrosine all have a generally similar shape.Consequently, the amino acids that comprise the followinggroups—arginine, lysine and histidine; alanine, glycine and serine; andphenylalanine, tryptophan and tyrosine—are defined herein asbiologically functional equivalents to the other amino acids in the samegroup. Other biologically functionally equivalent changes will beappreciated by those of skill in the art.

In another embodiment, the present invention provides isolated nucleicacids encoding functional fragments of the engineered enzymes of thepresent invention, or mutants thereof, in which conservativesubstitutions have been made for particular residues within the aminoacid sequence of any of the engineered sulfotransferase enzymesdescribed herein.

Additionally, isolated nucleic acids used to express arylsulfate-dependent sulfotransferases capable of practicing the methods ofthe present invention may be joined to other nucleic acid sequences foruse in various applications. Thus, for example, the isolated nucleicacids may be ligated into cloning or expression vectors, as are commonlyknown in the art and as described in the examples below. Additionally,nucleic acids may be joined in-frame to sequences encoding anotherpolypeptide so as to form a fusion protein, as is commonly known in theart. Fusion proteins can comprise a coding region for the arylsulfate-dependent sulfotransferase that is aligned within the sameexpression unit with other proteins or peptides having desiredfunctions, such as for solubility, purification, or immunodetection.Thus, in another embodiment, cloning, expression and fusion vectorscomprising any of the above-described nucleic acids, that encode for anaryl sulfate-dependent sulfotransferase that can be utilized in withmethods of the present invention are also provided.

Furthermore, nucleic acid segments of the present invention, regardlessof the length of the coding sequence itself, can be combined with otherDNA sequences, such as promoters, enhancers, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length can varyconsiderably. Those skilled in the art would recognize that a nucleicacid fragment of almost any length can be employed, with the totallength typically being limited by the ease of preparation and use in theintended recombinant DNA protocol.

In particular, recombinant vectors in which the coding portion of thegene or DNA segment is positioned under the control of a promoter areespecially useful. In some embodiments, the coding DNA segment can beassociated with promoters isolated from bacterial, viral, eukaryotic, ormammalian cells. Promoters specific to the cell type chosen forexpression are often the most effective. The use of promoter and celltype combinations for protein expression is generally known to those ofskill in the art of molecular biology (See, e.g., Sambrook et al. (2012)Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated byreference in its entirety). The promoters employed can be constitutiveor inducible and can be used under the appropriate conditions to directhigh-level expression of the introduced DNA segment, such as isadvantageous in the large-scale production of recombinant proteins orpeptides. Appropriate promoter systems that are often effective forhigh-level expression include, but are not limited to, the vacciniavirus promoter, the baculovirus promoter, and the Ptac promoter.

Thus, in some embodiments, an expression vector can be utilized thatcomprises a nucleotide sequence encoding for a biologically-active, arylsulfate-dependent sulfotransferase suitable for use with methods of thepresent invention. In one example, an expression vector can comprise anynucleotide sequence that encodes for an aryl sulfate-dependentsulfotransferase gene product. In further embodiments, an expressionvector comprises a nucleic acid comprising any of the nucleotidesequences described above, or any nucleotide sequence that encodes for apolypeptide comprising the amino acid sequence of any of the engineeredsulfotransferase enzymes described above. In even further embodiments,any nucleic acid sequence encoding for an engineered arylsulfate-dependent sulfotransferase enzyme of the present invention canbe codon-optimized based on the expression host used to produce theenzyme. The preparation of recombinant vectors and codon optimizationare well known to those of skill in the art and described in manyreferences, such as, for example, Sambrook et al. (2012) MolecularCloning: A Laboratory Manual, Fourth Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

Those skilled in the art would recognize that the DNA coding sequencesto be expressed, in this case those encoding the aryl sulfate-dependentsulfotransferase gene products, are positioned in a vector adjacent toand under the control of a promoter. As is known in the art, a promoteris a region of a DNA molecule typically within about 100 nucleotidepairs upstream of (i.e., 5′ to) the point at which transcription begins(i.e., a transcription start site). That region typically containsseveral types of DNA sequence elements that are located in similarrelative positions in different genes. It is understood in the art thatto bring a coding sequence under the control of such a promoter, onegenerally positions the 5′ end of the transcription initiation site ofthe transcriptional reading frame of the gene product to be expressedbetween about 1 and about 50 nucleotides “downstream” of (i.e., 3′ of)the chosen promoter.

One can also desire to incorporate into the transcriptional unit of thevector an appropriate polyadenylation site (e.g., 5′-AATAAA-3′), if onewas not contained within the original inserted DNA. Typically, poly-Aaddition sites are placed about 30 to 2000 nucleotides “downstream” ofthe coding sequence at a position prior to transcription termination.

Another type of discrete transcription regulatory sequence element is anenhancer. An enhancer imposes specificity of time, location andexpression level on a particular coding region or gene. A major functionof an enhancer is to increase the level of transcription of a codingsequence in a cell that contains one or more transcription factors thatbind to that enhancer. An enhancer can function when located at variabledistances from transcription start sites so long as a promoter ispresent.

Optionally, an expression vector of the invention comprises apolynucleotide operatively linked to an enhancer-promoter. As usedherein, the phrase “enhancer-promoter” means a composite unit thatcontains both enhancer and promoter elements. For example, an expressionvector can comprise a polynucleotide operatively linked to anenhancer-promoter that is a eukaryotic promoter and the expressionvector further comprises a polyadenylation signal that is positioned 3′of the carboxy-terminal amino acid and within a transcriptional unit ofthe encoded polypeptide. As used herein, the phrase “operatively linked”means that an enhancer-promoter is connected to a coding sequence insuch a way that the transcription of that coding sequence is controlledand regulated by that enhancer-promoter. Techniques for operativelylinking an enhancer-promoter to a coding sequence are well known in theart; the precise orientation and location relative to a coding sequenceof interest is dependent, inter alia, upon the specific nature of theenhancer-promoter.

An enhancer-promoter used in a vector construct of the present inventioncan be any enhancer-promoter that drives expression in a cell to betransfected. By employing an enhancer-promoter with well-knownproperties, the level and pattern of gene product expression can beoptimized.

Sulfotransferase enzymes suitable to practice the methods of the presentinvention can be expressed within cells or cell lines, eitherprokaryotic or eukaryotic, into which have been introduced the nucleicacids of the present invention so as to cause clonal propagation ofthose nucleic acids and/or expression of the proteins or peptidesencoded thereby. Such cells or cell lines are useful for propagating andproducing nucleic acids, as well as for producing the arylsulfate-dependent sulfotransferases themselves. As used herein, the term“transformed cell” is intended to embrace any cell, or the descendant ofany cell, into which has been introduced any of the nucleic acids of theinvention, whether by transformation, transfection, transduction,infection, or other means. Methods of producing appropriate vectors,transforming cells with those vectors, and identifying transformants arewell known in the art. (See, e.g., Sambrook et al. (2012) MolecularCloning: A Laboratory Manual, Fourth Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.)

Prokaryotic cells useful for producing transformed cells include membersof the bacterial genera Escherichia (e.g., E. coli), Pseudomonas (e.g.,P. aeruginosa), and Bacillus (e.g., B. subtilus, B. stearothermophilus),as well as many others well known and frequently used in the art.Prokaryotic cells are particularly useful for the production of largequantities of the proteins or peptides (e.g., aryl sulfate-dependentenzymes, fragments of those sequences thereof, or fusion proteinsincluding those sequences). Bacterial cells (e.g., E. coli) may be usedwith a variety of expression vector systems including, for example,plasmids with the T7 RNA polymerase/promoter system, bacteriophageregulatory sequences, or M13 Phage regulatory elements. Bacterial hostsmay also be transformed with fusion protein vectors that create, forexample, Protein A, lacZ, trpE, maltose-binding protein (MBP), smallubiquitin-related modifier (SUMO), poly-His tag, orglutathione-S-transferase (GST) fusion proteins. All of these, as wellas many other prokaryotic expression systems, are well known in the artand widely available commercially (e.g., pGEX-27 (Amrad, USA) for GSTfusions).

In some embodiments of the invention, expression vectors comprising anyof the nucleotide sequences described above can also comprise genes ornucleic acid sequences encoding for fusion proteins with any arylsulfate-dependent sulfotransferase. In further embodiments, expressionvectors can additionally include the malE gene, which encodes for themaltose binding protein. Upon inducing protein expression from suchexpression vectors, the expressed gene product comprises a fusionprotein that includes maltose binding protein and any of the arylsulfate-dependent sulfotransferase enzymes described above. In otherfurther embodiments, an expression vector that includes any of the abovenucleic acids that encode for any of the above aryl sulfate-dependentsulfotransferase enzymes can additionally include a gene encoding for aSUMO modifier, such as, in a non-limiting example, SUMO-1.

In other embodiments, expression vectors according to the presentinvention can additionally include a nucleic acid sequence encoding fora poly-His tag. Upon inducing protein expression from such expressionvectors, the expressed gene product comprises a fusion protein thatincludes the poly-His tag and any of the aryl sulfate-dependentsulfotransferase enzymes described above. In a further embodiment,expression vectors can include both a nucleic acid sequence encoding fora poly-His tag and the malE gene or a SUMO gene, from which a fusionprotein can be expressed that includes a poly-His tag, MBP, or SUMO,along with any aryl sulfate-dependent sulfotransferase enzyme.

Eukaryotic cells and cell lines useful for producing transformed cellsinclude mammalian cells (e.g., endothelial cells, mast cells, COS cells,CHO cells, fibroblasts, hybridomas, oocytes, embryonic stem cells),insect cells lines (e.g., Drosophila Schneider cells), yeast, and fungi.Non-limiting examples of such cells include, but are not limited to,COS-7 cells, CHO, cells, murine primary cardiac microvascularendothelial cells (CME), murine mast cell line C57.1, human primaryendothelial cells of umbilical vein (HUVEC), F9 embryonal carcinomacells, rat fat pad endothelial cells (RFPEC), and L cells (e.g., murineLTA tk− cells).

Vectors may be introduced into the recipient or “host” cells by variousmethods well known in the art including, but not limited to, calciumphosphate transfection, strontium phosphate transfection, DEAE dextrantransfection, electroporation, lipofection, microinjection, ballisticinsertion on micro-beads, protoplast fusion or, for viral or phagevectors, by infection with the recombinant virus or phage.

In another embodiment, the present invention provides arylsulfate-dependent sulfotransferase variants in which conservative ornon-conservative substitutions have been made for certain residueswithin any of the engineered sulfotransferase amino acid sequencesdisclosed above. Conservative or non-conservative substitutions can bemade at any point in the amino acid sequence, including residues thatsurround the active site or are involved in catalysis, provided that theenzyme retains measurable catalytic activity; namely, the transfer of asulfo group from an aryl sulfate compound to a polysaccharide,particularly a heparosan-based and/or HS polysaccharide. In otherembodiments, the aryl sulfate compound is PNS. In still otherembodiments, the aryl sulfate compound is NCS.

In another embodiment, the aryl sulfate-dependent sulfotransferaseenzymes have at least 50%, including at least 60%, 70%, 80%, 85%, 90% or95% up to at least 99% amino acid sequence identity to the amino acidsequence of any of the engineered sulfotransferase enzymes disclosedabove, including disclosed as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, and SEQ ID NOs: 33-54 and 56-61, while retainingits catalytic activity of transfer of a sulfo group from an aryl sulfatecompound to a polysaccharide, particularly a heparosan-based and/or HSpolysaccharide. Such sequences may be routinely produced by those ofordinary skill in the art, and sulfotransferase activity may be testedby routine methods such as those disclosed herein.

Further, and in another embodiment, the amino acid sequence(s) of any ofthe engineered aryl sulfate-dependent sulfotransferases utilized inaccordance with any of the methods described herein can be characterizedas a percent identity relative to a natural sulfotransferase thatcatalyzes the same reaction using PAPS as the sulfo donor, so long asthe sulfotransferase has aryl sulfate-dependent activity. For example,and in another embodiment, an engineered aryl sulfate-dependent NST thatcan be utilized in accordance with any of the methods of the presentinvention can comprise an amino acid sequence that has at least 50%,including at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, up toat least 97% sequence identity with the amino acid sequence of theN-sulfotransferase domain of any of the natural enzymes within the EC2.8.2.8 enzyme class, including biological functional fragments thereof.In a further embodiment, the engineered aryl sulfate-dependent NST cancomprise at least 50%, including at least 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95%, up to at least 97% sequence identity with the aminoacid sequence of the N-sulfotransferase domain of the natural humanglucosaminyl N-deacetylase/N-sulfotransferase enzyme (entrysp|P52848|NDST_1_HUMAN, in FIG. 3 , above).

In another embodiment, an engineered aryl sulfate-dependent 2OST thatcan be utilized in accordance with any of the methods of the presentinvention can comprise an amino acid sequence that has at least 50%,including at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, up toat least 97% sequence identity with the amino acid sequence of any ofthe natural 2OST enzymes within the EC 2.8.2.- enzyme class, includingbiological functional fragments thereof. In a further embodiment, theengineered aryl sulfate-dependent 2OST can comprise at least 50%,including at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, up toat least 97% sequence identity with the amino acid sequence of thenatural chicken 2OST enzyme (entry sp|Q76KB1|HS2ST_CHICK, in FIGS.14A-14D, above).

In another embodiment, an engineered aryl sulfate-dependent 6OST thatcan be utilized in accordance with any of the methods of the presentinvention can comprise an amino acid sequence that has at least 50%,including at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, up toat least 97% sequence identity with the amino acid sequence of any ofthe natural 6OST enzymes within the EC 2.8.2.- enzyme class, includingbiological functional fragments thereof. In a further embodiment, theengineered aryl sulfate-dependent 6OST can comprise at least 50%,including at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, up toat least 97% sequence identity with the amino acid sequence of the firstisoform of the mouse 6OST (UniProtKB Accession No. Q9QYK5). In a furtherembodiment, the engineered aryl sulfate-dependent 6OST can comprise atleast 50%, including at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or95%, up to at least 97% sequence identity with residues 67-377 of theamino acid sequence of the first isoform of the mouse 6OST (entryQ9QYK5|H6ST1_MOUSE, in FIGS. 18A-18C, above).

In another embodiment, an engineered aryl sulfate-dependent 3OST thatcan be utilized in accordance with any of the methods of the presentinvention can comprise an amino acid sequence that has at least 50%,including at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, up toat least 97% sequence identity with the amino acid sequence of any ofthe natural enzymes within the EC 2.8.2.23 enzyme class, includingbiological functional fragments thereof. In a further embodiment, theengineered aryl sulfate-dependent 3OST can comprise at least 50%,including at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, up toat least 97% sequence identity with residues 48-311 of the amino acidsequence of the first isoform of the natural human 3OST (UniProtKBAccession No. O14792).

Substantially pure aryl sulfate-dependent sulfotransferases may bejoined to other polypeptide sequences for use in various applications.Thus, for example, engineered sulfotransferases may be joined to one ormore additional polypeptides so as to form a fusion protein, as iscommonly known in the art. The additional polypeptides may be joined tothe N-terminus, C-terminus or both termini of the aryl sulfate-dependentsulfotransferase enzyme. Such fusion proteins may be particularly usefulif the additional polypeptide sequences are easily identified (e.g., byproviding an antigenic determinant), are easily purified (e.g., byproviding a ligand for affinity purification), or enhance the solubilityof the aryl sulfate-dependent sulfotransferase enzyme in solution.

In another embodiment, substantially pure proteins may comprise only aportion or fragment of the amino acid sequence of a complete arylsulfate-dependent sulfotransferase. In some instances, it may bepreferable to employ a minimal fragment retaining aryl sulfate-dependentsulfotransferase activity, particularly if the minimal fragment enhancesthe solubility or reactivity of the enzyme. Thus, in some embodiments,methods of the present invention can be practiced using substantiallypure aryl sulfate-dependent sulfotransferases of any length, includingfull-length forms, or minimal functional fragments thereof.Additionally, these proteins may also comprise conservative ornon-conservative substitution variants as described above.

In some embodiments, the present invention provides substantially purepreparations of aryl sulfate-dependent sulfotransferases, includingthose comprising any of the amino acid sequences disclosed above. Theengineered sulfotransferases may be substantially purified by any of avariety of methods selected on the basis of the properties revealed bytheir protein sequences. Typically, the aryl sulfate-dependentsulfotransferases, fusion proteins, or fragments thereof, can bepurified from cells transformed or transfected with expression vectors,as described above. Insect, yeast, eukaryotic, or prokaryotic expressionsystems can be used, and are well known in the art. In the event thatthe protein or fragment localizes within microsomes derived from theGolgi apparatus, endoplasmic reticulum, or other membrane-containingstructures of such cells, the protein may be purified from theappropriate cell fraction. Alternatively, if the protein does notlocalize within these structures, or aggregates in inclusion bodieswithin the recombinant cells (e.g., prokaryotic cells), the protein maybe purified from whole lysed cells or from solubilized inclusion bodiesby standard means.

Purification can be achieved using standard protein purificationprocedures including, but not limited to, affinity chromatography,gel-filtration chromatography, ion-exchange chromatography,high-performance liquid chromatography (RP-HPLC, ion-exchange HPLC,size-exclusion HPLC), high-performance chromatofocusing chromatography,hydrophobic interaction chromatography, immunoprecipitation, orimmunoaffinity purification. Gel electrophoresis (e.g., PAGE, SDS-PAGE)can also be used to isolate a protein or peptide based on its molecularweight, charge properties and hydrophobicity.

An aryl sulfate-dependent sulfotransferase, or a fragment thereof, mayalso be conveniently purified by creating a fusion protein including thedesired sequence fused to another peptide such as an antigenicdeterminant, a poly-histidine tag (e.g., QIAexpress vectors, QIAGENCorp., Chatsworth, Calif.), or a larger protein (e.g., GST using thepGEX-27 vector (Amrad, USA), green fluorescent protein using the GreenLantern vector (GlBCO/BRL. Gaithersburg, Md.), maltose binding proteinusing the pMAL vector (New England Biolabs, Ipswich, Mass.), or a SUMOprotein. The fusion protein may be expressed and recovered fromprokaryotic or eukaryotic cells and purified by any standard methodbased upon the fusion vector sequence. For example, the fusion proteinmay be purified by immunoaffinity or immunoprecipitation with anantibody to the non-aryl sulfate-dependent sulfotransferase portion ofthe fusion or, in the case of a poly-His tag, by affinity binding to anickel column. The desired aryl sulfate-dependent sulfotransferaseprotein or fragment can then be further purified from the fusion proteinby enzymatic cleavage of the fusion protein. Methods for preparing andusing such fusion constructs for the purification of proteins are wellknown in the art and numerous kits are now commercially available forthis purpose.

Furthermore, in some embodiments, isolated nucleic acids encoding forany aryl sulfate-dependent sulfotransferase may be used to transformhost cells. The resulting proteins may then be substantially purified bywell-known methods including, but not limited to, those described in theexamples below. Alternatively, isolated nucleic acids may be utilized incell-free in vitro translation systems. Such systems are also well knownin the art.

While particular embodiments of the invention have been described, theinvention can be further modified within the spirit and scope of thisdisclosure. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, numerousequivalents to the specific procedures, embodiments, claims, andexamples described herein. As such, such equivalents are considered tobe within the scope of the invention, and this application is thereforeintended to cover any variations, uses or adaptations of the inventionusing its general principles. Further, the invention is intended tocover such departures from the present disclosure as come within knownor customary practice in the art to which this invention pertains andwhich fall within the appended claims.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

The contents of all references, patents, and patent applicationsmentioned in this specification are hereby incorporated by reference,and shall not be construed as an admission that such reference isavailable as prior art to the present invention. All of the incorporatedpublications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains, and are incorporated to the same extent as if eachindividual publication or patent application was specifically indicatedand individually indicated by reference.

The invention is further illustrated by the following working andprophetic examples, neither of which should be construed as limiting theinvention. Additionally, to the extent that section headings are used,they should not be construed as necessarily limiting. Any use of thepast tense to describe an example otherwise indicated as constructive orprophetic is not intended to reflect that the constructive or propheticexample has actually been carried out.

EXAMPLES

The following working and prophetic examples illustrate the embodimentsof the invention that are presently best known. However, it is to beunderstood that the following are only exemplary or illustrative of theapplication of the principles of the present invention. Numerousmodifications and alternative compositions, methods, and systems may bedevised by those skilled in the art without departing from the spiritand scope of the present invention. Thus, while the present inventionhas been described above with particularity, the following examplesprovide further detail in connection with what are presently deemed tobe the most practical and preferred embodiments of the invention.

Example 1: Cloning, Expression, and Purification of the Engineered ArylSulfate-Dependent Sulfotransferases

A study was conducted in accordance with embodiments of the presentdisclosure to determine whether genes according to the present inventioncould be transformed into host cells capable of overexpressingengineered aryl sulfate-dependent sulfotransferases. After expression,each aryl sulfate-dependent enzyme was isolated and purified from thehost cell.

Generally, DNA coding for genes of any sequence can be synthesized denovo by methods commonly known in the art, including but not limited tooligonucleotide synthesis and annealing. Alternatively, DNA can besynthesized commercially and purchased from any one of severallaboratories that regularly synthesize genes of a given sequence,including but not limited to ThermoFisher Scientific, GenScript, DNA2.0, or OriGene. Persons skilled in the art would appreciate that thereare several companies that provide the same services, and that the listprovided above is merely a small sample of them. Genes of interest canbe synthesized independently and subsequently inserted into a bacterialor other expression vector using conventional molecular biologytechniques, or the genes can be synthesized concurrently with the DNAcomprising the expression vector itself. Similar to genes of interest,suitable expression vectors can also be synthesized or obtainedcommercially. Often, bacterial expression vectors include genes thatconfer selective antibiotic resistance to the host cell, as well asgenes that permit the cell to overproduce the protein of interest inresponse to the addition of isopropyl β-D-1-thiogalactopyranoside(IPTG). Bacterial production of proteins of interest using IPTG toinduce protein expression is widely known in the art.

As described above, expression vectors can also include genes thatenable production of fusion proteins that include the desired proteinthat is co-expressed with an additional, known protein to aid in proteinfolding and solubility. Non-limiting examples of fusion proteins thatare commonly produced and are well-known in the art include fusions withMBP, SUMO, or green fluorescent protein. In particular, MBP fusionproteins facilitate easier purification because MBP possesses highaffinity for amylose-based resins used in some affinity chromatographycolumns, while SUMO fusion proteins can include a poly-histidine tagthat enables affinity purification on columns with Ni²⁺-based resins asa stationary phase. Often, fusion proteins between the protein ofinterest and MBP and/or SUMO can optionally include an amino acidlinking sequence that connects the two proteins. Non-limiting examplesof commercial expression vectors that can be purchased to produce MBPfusion proteins include the pMAL-c5E™ and pMAL-c5X™ vectors, which canbe obtained from New England Biolabs. Similarly, and in anothernon-limiting example, commercial expression vectors can also bepurchased to produce SUMO fusion proteins, such as the pE-SUMOpro AMPvector, available from LifeSensors, Inc. Once the fusion proteins areproduced and isolated, proteases can be utilized to cleave the fusedprotein and any associated linker sequences from the sulfotransferase,if cleavage is necessary for activity.

Additionally, expression vectors can also include DNA coding for apoly-histidine tag that can be synthesized at either the N- orC-terminus of the protein of interest. As with MBP fusions, proteinsthat include a poly-histidine tag simplify the enzyme purificationbecause the tag has a high affinity for Ni²⁺ resins that are utilized inmany purification columns. Additionally, poly-histidine tags canoptionally be cleaved after purification if it is necessary for optimalactivity of the enzyme. A non-limiting example of an expression vectorencoding for a C-terminal poly-histidine tag is the pET21b vector,available from Novagen. Another non-limiting example of an expressionvector encoding for a poly-histidine tag is the pE-SUMO vector, whichencodes for a poly-histidine tag at the N-terminus of the SUMO protein.

In the present example, double-stranded DNA fragments comprising thenucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, orSEQ ID NO: 27, encoding for engineered aryl sulfate-dependentsulfotransferases comprising the amino acid sequences of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO:22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, respectively, weresynthesized using Integrated DNA Technologies' (IDT) gBlocks® GeneFragments synthesis service. Polymerase chain reactions (PCR) wereinitiated to generate copies of each double-stranded DNA fragment, usingforward and reverse primers comprising appropriate restriction enzymerecognition sequences to facilitate insertion into an expression vector.Genes encoding for the engineered NST enzymes (SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11) and 3OSTenzymes (SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27) contained NdeI andBamHI restriction enzyme recognition sequences, and were ligated intothe pMAL-c5x expression vector using quick ligation kits provided byNEB. Expression vectors were then transformed into competent DH5-α E.coli cells. Single clones were incubated in LB medium with 100 μL/mLampicillin. Nucleotide sequences of each gene and expression vectorwithin the transformed host cells were confirmed by commercial DNAsequencing (GeneWiz).

Protein expression of the glucosaminyl N- and 3-O sulfotransferaseenzymes was achieved by first transforming confirmed DNA constructs intocompetent SHuffle® T7 Express lysY E. coli cells. Protein expression ofthe glucosaminyl N- and 3-O sulfotransferase enzymes has also beenachieved by transforming confirmed DNA constructs into competent BL21(DE3) E. coli cells. From either construct, resultant colonies were usedto inoculate 250 mL cultures in LB medium, which were allowed to shakeand incubate at 32° C. until an optical density at 600 nM (OD 600) ofapproximately 0.4 to 0.6 was observed. Expression was induced by theaddition of 100 μM IPTG to each culture at 18° C.

Upon incubation at 18° C. overnight, expressed cells were harvested bycentrifuging at 3,620 g and resuspending the pellet in 10 mL ofresuspension buffer (25 mM Tris-HCl, pH 7.5; 0.15 M NaCl; 0.2 mg/mLlysozyme; 10 μg/ml DNase I; 5 mM MgCl₂; and 0.1% (w/v) Triton-X 100).Resuspended cells were lysed upon sonication on ice for three pulses of10 seconds each, and subsequently passed through a 0.45-μm syringefilter. The resulting supernatant was loaded into a 5-mL spin column(G-biosciences) comprising Dextrin Sepharose® resin (GE Biosciences)suspended in a binding buffer comprising 25 mM Tris-HCl, pH 7.5 and 0.15M NaCl. Enzymes of interest were eluted from the column upon adding anelution buffer comprising 25 mM Tris-HCl, pH 7.5; 0.15 M NaCl; and 40 mMmaltose.

On the other hand, genes encoding for the engineered 2OST (SEQ ID NO:13, SEQ ID NO: 15) and 6OST enzymes (SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21) contained BsaI and XbaI restriction enzyme recognitionsequences, and were ligated into the pE-SUMO vector (LifeSensors, Inc.).Expression vectors were then transformed into competent BL21-DE3 E. colicells. Single clones were incubated in Terrific Broth with 100 μL/mLampicillin. Nucleotide sequences of each gene and expression vectorwithin the transformed host cells were confirmed by commercial DNAsequencing (GeneWiz).

Protein expression of the engineered 2OSTs and 6OSTs was achieved byinoculating 500 mL cultures in Terrific Broth with ampicillin andallowing the cultures to incubate with shaking at 35° C. until an OD 600of approximately 0.6-0.8 was reached. Protein expression was induced bythe addition of 0.2 mM IPTG at 18° C. Cultures were then allowed toincubate at 18° C. overnight, and were subsequently lysed and filteredusing an identical procedure to the glucosaminyl N- and 3-Osulfotransferase enzymes above. The 2OST and 6OST enzymes weresubsequently purified in a 5-mL spin column (G-biosciences) comprisingHisPur Ni-NTA resin (Thermofisher) suspended in a binding buffercomprising 25 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 5 mM MgCl₂, and 30 mMimidazole. Enzymes of interest were eluted from the column upon addingan elution buffer comprising 25 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 5 mMMgCl₂, and 300 mM imidazole.

Example 2: Mass Spectrometric Characterization of the N-SulfatedPolysaccharide Products of Engineered Aryl Sulfate-Dependent NST Enzymes

A study was conducted in accordance with embodiments of the presentdisclosure to confirm NST activity of enzymes comprising the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, or SEQ ID NO: 12 by detecting the presence of N-sulfatedpolysaccharide products formed as a result of their sulfotransferreaction, using mass spectrometry (MS). Each engineered enzyme waspurified according to the procedure of Example 1. Sulfotransferaseactivity was monitored in 100 μL reactions containing 50 μM of enzyme.To each purified protein solution, 20 mg of an aryl sulfate compound(either PNS or NCS) was dissolved in 2 mL of reaction buffer (50 mMIVIES pH 7.0, 2 mM CaCl₂)), added to the protein solution, and incubatedat 37° C. for 10 min. 2.5 mL of 2 mg/mL solution of N-deacetylatedheparosan was added to protein/donor solution and incubated overnight at37° C. The N-deacetylated heparosan was synthesized according to theprotocol described in Balagurunathan, K. et al (eds.) (2015),Glycosaminoglycans: Chemistry and Biology, Methods in Molecular Biology,vol. 1229, DOI 10.1007/978-1-4939-1714-3_2, ©Springer Science+BusinessMedia, New York, pp. 11-19 (section 3.1). To purify the N-sulfatedproduct, the incubated reaction mixture was centrifuged the followingday at 5,000×g for 10 min. The filter was washed once with 2 mL water,and centrifuged again. The filtrate was added to a 1K MWCO Dialysismembrane, dialyzed for 2 days in Milli-Q water, with water changes at 1h, 2 h, 8 h, 16 h, 32 h, and then lyophilized.

The lyophilized N-sulfated products from each reaction were subsequentlydigested with a mixture of three carbon-oxygen lyases comprising theamino acid sequences of SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32,which catalyze the β-eliminative cleavage of heparosan-basedpolysaccharides. Such lyases are available from New England Biolabs,among other chemical and biological commercial entities. 1 μL of eachlyase was incubated with 50 μg of the lyophilized sulfatedpolysaccharide product and the provided digestion buffer, and incubatedover 24 hours according to the packaged instructions provided by NewEngland Biolabs with each lyase. After digestion, the lyase enzymes wereinactivated by heating to 100° C. for 5 minutes. Samples werecentrifuged at 14,000 rpm for 30 minutes before introduction to a stronganion exchange, high performance liquid chromatography (SAX) analysis.SAX analysis was performed on a Dionex Ultimate 3000 LC systeminterface. Separation was carried out on a 4.6×250 mm Waters Spherisorbanalytical column with 5.0 μm particle size at 45° C. Mobile phasesolution A was 2.5 mM sodium phosphate, pH 3.5, while mobile phasesolution B was 2.5 mM sodium phosphate, pH 3.5, and 1.2 M Sodiumperchlorate. After each sample was loaded onto the column, mobile phasesolutions were applied to the column at a ratio of 98% mobile phasesolution A and 2% mobile phase solution B for five minutes at a flowrate of 1.4 mL/min. After five minutes, a linear gradient of increasingmobile phase solution B was applied until the ratio of mobile phasesolution A to mobile phase solution B was 50:50.

Using the SAX analysis, it was determined that all six of the testedenzymes were active as sulfotransferases. However, each of thesulfotransferases were not necessarily active with both PNS and NCS.Enzymes having the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4,and SEQ ID NO: 10 had activity with NCS only, and the enzyme having theamino acid sequence of SEQ ID NO: 12 had activity with PNS only. Enzymeshaving the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 8 hadactivity with both aryl sulfate compounds.

Representative chromatograms from SAX analysis illustrating the presenceof N-sulfated products produced as a result of the reaction are shown inFIG. 26 . Both the starting material and product were digested with thelyases having the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31,and SEQ ID NO: 32 according the digestion procedure described above. Twodisaccharide standards (HD005 and HD013) that are commercially availablefrom Iduron, Ltd were also analyzed using SAX. The HD013 disaccharidecomprises an unsubstituted glucosamine residue and a reduced hexuronicacid. The HD005 disaccharide is the same as HD013 except that theglucosamine residue is N-sulfated. All of the overlaid chromatograms arenormalized so the most prominent peak in each chromatogram is assigned anormalized relative fluorescence value of 1.0.

As shown in FIG. 26 , the most prominent peak for HD013 disaccharide(illustrated with a * symbol) elutes almost immediately, whereas themost prominent peak for the HD005 disaccharide (illustrated with a **symbol) elutes after approximately 17 minutes. This is expected underSAX conditions because positively-charged species (like HD013) typicallydo not bind to the column, whereas negatively-charged species (likeHD005) do bind to the column. The N-deacetylated heparosan, which issimilarly non-sulfated, most prominently elutes at a nearly identicaltime as HD013. Similarly, the lyophilized sample produced during thereaction shows a peak at a nearly identical time as HD005, indicatingthat the sample likely contains an N-sulfated product. Other peakswithin each of the chromatograms, particularly within the synthesizedstarting materials and products, indicate a lack of sample purity basedon the use of spin-filtration columns as the sole basis of purifying thepolysaccharides in each instance. Those skilled in the art wouldappreciate that there are several other separations techniques that canbe utilized if a more purified product is desired. Additionally, thedrifting upward of the baseline of the fluorescent signal in thechromatograms is a known phenomenon when increasing amounts of salt areintroduced onto the column via the mobile phase.

Example 3: Mass Spectrometric Characterization of the 2-O SulfatedPolysaccharide Products of Engineered Aryl Sulfate-Dependent 2OSTEnzymes

A study was conducted in accordance with embodiments of the presentdisclosure to confirm 2OST activity of enzymes comprising the amino acidsequence of SEQ ID NO: 14 or SEQ ID NO: 16 by detecting the presence of2-O sulfated polysaccharide products formed as a result of theirsulfotransfer reaction, using a similar procedure as in Example 2,except that the sulfo acceptor polysaccharide was commercial heparin inwhich the 2-O sulfate groups had been selectively removed by chemicalmeans (product DSH001/2, available from Galen Laboratory Supplies) andanalysis of each of the digested samples containing sulfated productswas conducted using mass spectrometry, coupled with SAX-based highperformance liquid chromatography (LCMS).

Disaccharides obtained by digesting the 2-O sulfated products using thecarbon-oxygen lyases having the amino acid sequence of SEQ ID NO: 30,SEQ ID NO: 31, and SEQ ID NO: 32 and according to the proceduredescribed above in Example 2 were quantified on a Shimadzu LCMS-8050Triple Quadrupole Liquid Chromatograph Mass Spectrometer. 100 ng of eachof the digested samples, diluted in 10 mM ammonium bicarbonate (pH 10).The disaccharides were separated on a Thermo Hypercarb HPLC column(100×2.1 mm, 5 μm). The mobile phase consisted of 10 mM ammoniumbicarbonate (pH 10), and the disaccharides were eluted with anacetonitrile gradient of 0% to 20% for 2.5 min, held at 20% for the next2.5 min, with 2 min of equilibration at 0% before the next injection;the flow rate was 0.2 mL/min, and the total run time was 7.1 min.

The extracted ion chromatograms from the LCMS are shown in FIGS. 27A and27B, corresponding to 2-O sulfated products obtained from reactions withengineered enzymes having the amino acid sequences of SEQ ID NO: 14 orSEQ ID NO: 16. Peaks were compared with chromatograms of a series ofeight disaccharide standards, as well as a chromatogram from 100 ng of acommercial heparin polysaccharide (CAS code: 9041-08-1, available fromMillipore Sigma), which was also digested using the lyase mixture. Theeight reference disaccharide standards (D0A0, D0S0, D0A6, D2A0, D0S6,D2S0, D2A6, D2S6) represent disaccharides that are variably sulfated atthe N-, 2-O and 6-O positions. In particular, the disaccharide D2S0represents a disaccharide having a hexuronyl residue sulfated at the 2-Oposition and an N-sulfated glucosamine residue. The retention time andpeak areas from the spectra from all of the disaccharide standards (notshown), the digested commercial sulfated polysaccharide (not shown), andthe sulfated polysaccharide products of the engineered enzymes havingthe amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16 are collectedin Table 1, below. Since the ionization of each individual disaccharideis different, the present percent in EIC chromatograms may not representtheir actual abundance. However, the ionization efficiency is identicalfor each disaccharide from sample to sample. Therefore, it is believedthat comparing the peak area percent of the same saccharides from sampleto sample can still be achieved.

TABLE 1 Peak Area % Peak Commercial SEQ ID SEQ ID No. Disaccharidesstandard NO: 14 NO: 16 1 D0A0 3.9 5.9 9.1 2 D0S0 3.9 87.1  85.5  3 D0A63.4 ND ND 4 D2A0 1.8 ND ND 5 D0S6 11.8  4.1 3.1 6 D2S0 6.6 2.9 2.3 7D2A6 1.6 ND ND 8 D2S6 67.0  ND ND

Sulfotransferase activity of the engineered enzymes was confirmed by there-sulfation at the 2-O position of hexuronic acid residues within thesulfo acceptor polysaccharide that had previously been desulfated priorto the reaction. This is illustrated by the presence of D2S0disaccharides within the products isolated from reactions of bothengineered enzymes and NCS. Without being limited by a particulartheory, it is also believed that the activity of the engineered enzymeis dependent on reacting with a portion of the polysaccharide in whichthe hexuronic acid residue is adjacent to a glucosamine residue that isN-sulfated, but not 6-O sulfated. This is illustrated by the lack ofD2S6 (2-O sulfated hexuronic acid residue and an N,6-sulfatedglucosamine residue) and D2A6 (2-O sulfated hexuronic acid residue and a6-O sulfated N-acetyl glucosamine residue) disaccharides detected withinthe isolated sulfated polysaccharide product. This is a similarreactivity to wild type 2OSTs within EC 2.8.2.-, which are believed toreact with N-sulfated heparosan comprising either the structure ofFormula IV or Formula V.

Example 4: Mass Spectrometric Characterization of the 6-O SulfatedPolysaccharide Products of Engineered Aryl Sulfate-Dependent 6OSTEnzymes

A study was conducted in accordance with embodiments of the presentdisclosure to confirm 6OST activity of enzymes comprising the amino acidsequence of SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22 by detectingthe presence of 6-O sulfated polysaccharide products as a result oftheir sulfotransfer reaction, using a similar LCMS procedure as inExample 3, except that the sulfo acceptor polysaccharide was prepared bychemically 6-O desulfating commercially available heparin (CAS code:9041-08-1, available from Millipore Sigma), according to the procedureprovided by Kariya, Y., et al., (2000) J. Biol. Chem. 275(34):25949-25958).

The extracted ion chromatograms corresponding to 6-O sulfated productsobtained from reactions with engineered enzymes having the amino acidsequences of SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22 are shown inFIG. 28A, FIG. 28B, and FIG. 28C, respectively. Enzymes having thesequence of SEQ ID NO: 18 and SEQ ID NO: 20 were active when NCS was thesulfo group donor, while the enzyme having the sequence of SEQ ID NO: 22was active when PNS was the sulfo group donor. Assigned peaks were basedon the determined retention times of eight reference disaccharidestandards. The eight reference disaccharide standards (D0A0, D0S0, D0A6,D2A0, D0S6, D2S0, D2A6, and D2S6) represent disaccharides that arevariably sulfated at the N-, 2-O, and 6-O positions. D0A6, D0S6, D2A6,and D2S6 comprise 6-O sulfated glucosamine residues. S6 indicates anN,6-sulfated glucosamine residue, while A6 indicates a 6-O sulfatedN-acetyl glucosamine residue. Each chromatogram indicates two integrablepeaks, D0S6 and D2S6, correlating to the synthesis of N,6-sulfatedglucosamine residues, adjacent to a hexuronic acid residue that iseither non sulfated or sulfated at the 2-O position, respectively. Thepeak area % of all the labelled disaccharides is in Table 2, below.Since the ionization of each individual disaccharide is different,especially for D0A0 and D2S6, the present percent in EIC chromatogramsmay not represent their actual abundance. However, the ionizationefficiency is identical for each disaccharide from sample to sample.Therefore, it is believed that comparing the peak area percent of thesame saccharides from sample to sample can still be achieved.

TABLE 2 Peak Area % Peak RT SEQ ID SEQ ID SEQ ID No. Disaccharides (min)NO: 18 NO: 20 NO: 22 1 D0A0 7.7 4.6 6.0 5.4 2 D0S0 16.4  14.2  18.4 13.0  3 D0A6 ND ND ND ND 4 D2A0 20.0  1.1 1.8 1.3 5 D0S6 23.7  4.0 3.75.6 6 D2S0 25.6  73.5  68.4  72.4  7 D2A6 ND ND ND ND 8 D2S6 32.7  2.51.7 2.3

Sulfotransferase activity of the engineered enzymes was confirmed by there-sulfation at the 6-O position of glucosamine residues that had beendesulfated by the procedure according to Kariya, Y., et al, above. Thisis illustrated by the presence of DOSE and D2S6 disaccharides within theproducts isolated from the reactions with each enzyme. Among each of theengineered enzymes, it appears that the 6OST having the amino acidsequence of SEQ ID NO: 22 was the most active, based on comparing thepeak area percentages of the D0S6 and D2S6 disaccharides. However, whileD0A6 and D2A6 polysaccharides were not observed in any of the 6-Osulfated products produced by the engineered enzymes, without beinglimited by any particular theory, it is believed that these enzymes maynonetheless be able to transfer a sulfo group to N-acetyl glucosamineresidues in different reaction conditions, particularly by increasingthe concentration of the enzyme and/or polysaccharide where the presenceof N-acetyl glucosamine residues is confirmed prior to the reaction,based on the reactivity of natural natural 6OSTs within EC 2.8.2.-.

Example 5: Mass Spectrometric Characterization of the 3-O SulfatedPolysaccharide Products of Engineered Aryl Sulfate-Dependent 3OSTEnzymes

A study was conducted in accordance with embodiments of the presentdisclosure to confirm 3OST activity of enzymes comprising the amino acidsequence of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28 by detectingthe presence of 3-O sulfated polysaccharide products as a result oftheir sulfotransfer reaction, using a reaction, using a similar LCMSprocedure as in Example 3, except that the sulfo acceptor polysaccharidewas commercially-available heparin (CAS code: 9041-08-1, available fromMillipore Sigma). Even though the unmodified heparin contains ˜3.5%(w/w) of 3-O sulfated glucosamine residues, about ˜60% of theglucosamine residues are N,6-sulfated and are adjacent to a 2-O sulfatedhexuronic acid residue, as in Formula X. Consequently, theseN,6-sulfated glucosamine residues can still be 3-O sulfated.

The extracted ion chromatograms are shown in FIG. 29A and FIG. 29B,along with chromatograms of a series of ten reference standards and 100ng of the commercial polysaccharide, which was also digested using thelyase mixture. The ten reference standards (D0A0, D0S0, D0A6, D2A0,DOSE, D2S0, D2A6, D2S6, D0A6G0S3, and D0A6G0S9) represent di- ortetrasaccharides that are variably sulfated at the N-, 2-O, 3-O, and 6-Opositions (black spectrum). For clarity, reference peaks that include3-O sulfated glucosamine residues (D0A6G0S3) and (D0A6G0S9) areindicated in the digested commercial polysaccharide spectrum, shown inred. Four mass spectra representing the digested sulfated polysaccharideproducts from reactions with enzymes comprising the amino acid sequenceof SEQ ID NO: 24 (PNS, yellow spectrum), SEQ ID NO: 26 (PNS, purplespectrum) (NC S, green spectrum), and SEQ ID NO: 28 (NCS, blue spectrum)are shown below the digested commercial polysaccharide spectrum. Thepeak area % of all the labelled disaccharides and tetrasaccharides is inTable 3, below. Since the ionization of each individual disaccharide isdifferent, especially for D0A0 and D2S6, the present percent in EICchromatograms may not represent their actual abundance. However, theionization efficiency is identical for each disaccharide ortetrasaccharide from sample to sample. Therefore, it is believed thatcomparing the peak area percent of the same saccharides from sample tosample can still be achieved.

TABLE 3 Peak Area % SEQ ID SEQ ID peak RT Commercial SEQ ID NO: 26 SEQID NO: 26 No. Disaccharides (min) standard NO: 24 (NCS) NO: 28 (PNS) 1D0A0 4.5 1.9 0.6 0.8 1.4 N.D. 2 D0S0 22.5 3.7 1.4 1.7 2.3 N.D. 3 D0A624.6 4.2 2.8 3.1 4.5 N.D. 4 D2A0 26.2 2.2 0.5 0.8 0.5 N.D. 5 D0S6 37.516.0 10.9 10.6 13.1 N.D. 6 D2S0 38.5 6.5 4.9 5.4 5.4 N.D. 7 D2A6 40.31.6 0.8 0.8 0.9 N.D. 8 D2S6 48.4 60.3 73.4 71.6 64.0 100.0 9 D0A6G0S352.9 0.6 0.8 0.9 1.4 N.D. 10 D0A6G0S9 58.2 3.0 4.0 4.1 6.5 N.D.

Sulfotransferase activity of each of the engineered enzymes wasconfirmed by the increase in the abundance of the D0A6G0S3 (hexuronicacid-6-O-sulfated N-acetyl glucosamine-glucuronic acid-N,3,6-sulfatedglucosamine) and D0A6G0S9 (hexuronic acid-6-O-sulfated N-acetylglucosamine-glucuronic acid-N,3-sulfated glucosamine) tetrasaccharidesrelative to the commercial heparin sample. However, the total abundanceof disaccharides in the SEQ ID NO: 26 PNS sample was much lower thanother samples. Subsequent trials included re-running the experiment with10 times more injection volume, and a re-digestion of the sample withthe lyase mixture. Nonetheless, only the D2S6 disaccharide could ever befound, indicating that the abundance of the SEQ ID NO: 26 PNS sulfatedpolysaccharide sample isolated initially was extremely low, and/or thatthe polysaccharide resists lyase digestion, causing the product topotentially elute from the column with a retention time longer than onehour.

Nonetheless, NMR studies (indicated below in Example 6) indicated 3-Osulfotransferase activity with the enzyme comprising the amino acidsequence SEQ ID NO: 26 when PNS is the aryl sulfate compound. Further,the enzyme having the amino acid sequence of SEQ ID NO: 26 wasdetermined to be active as a sulfotransferase when NCS is the arylsulfate compound. Therefore, it is believed that the observed resultsfor the SEQ ID NO: 26 PNS sulfated polysaccharide sample during the LCMSexperiment result from the sample produced for the purpose of theexperiment, and not the activity of the enzyme itself. Otherwise, ahigher abundance of 3-O sulfation was found in all of the other sulfatedpolysaccharide products from SEQ ID NO: 24, SEQ ID NO: 26, and SEQ IDNO: 28, relative to the commercial heparin standard.

Example 6: Confirmation of Sulfotransferase Activity of the Engineered3OSTs Using Nuclear Magnetic Resonance

A study was conducted in accordance with embodiments of the presentdisclosure to confirm the 3-O sulfotransferase activity of theengineered enzymes having the amino acid sequence of SEQ ID NO: 24, SEQID NO: 26, and SEQ ID NO: 28, particularly the activity of the enzymehaving the amino acid sequence SEQ ID NO: 26 with PNS as the sulfo groupdonor. Each enzyme was purified according to the procedure of Example 1.To each purified protein solution, 20 mg of an aryl sulfate compound(PNS or NCS) dissolved in 2 mL of reaction buffer (50 mM MES pH 7.0, 2mM CaCl₂)) was added to the protein solution and incubated at 37° C. for10 min. 2.5 mL of 2 mg/mL solution of the commercial heparinpolysaccharide utilized in Example 5 was added to protein/donor solutionand incubated overnight at 37° C.

Each reaction was centrifuged at 5,000×g for 10 min, applied to apre-wetted 30K MWCO Amicon-15 filter and centrifuged at 5,000×g for 10min. The filter was washed once with 2 mL water, and centrifuged again.The filtrate was added to a 1K MWCO Dialysis membrane, dialyzed for 2days in Milli-Q water, with water changes at 1 h, 2 h, 8 h, 16 h, 32 h,and then lyophilized. The dry, white powder was resuspended in 400 μLD20, lyophilized to remove exchangeable protons, then resuspended in 600μL D₂O and transferred to NMR tubes (Wilmad, 0.38 mm×7″). To determineif sulfotransfer took place, ¹H NMR spectra were obtained on a Bruker600 MHz NMR, 32 scans, with water suppression. The overall reactionscheme is shown in FIG. 30 . Within FIG. 30 , the 3-O positions of anyof the glucosamine residues can be sulfated by the 3OST enzyme. Thesulfated 3-O position is circled in the central polysaccharide.Exchangeable protons having the ability to exhibit resonance upondeuterium exchange are shown in bold, in the bottom polysaccharide.Crude mixture peaks were integrated to literature-referenced spectra forthe sulfo acceptor polysaccharide and associated 3-O sulfated product.

As shown in the overlain spectra in FIG. 31 , a sharp peak at 5.15 ppmthat correlates to the proton at the C2 carbon of the 2-O sulfatediduronic acid present in the commercial heparin disappears upon reactingwith enzymes comprising the amino acid sequence of SEQ ID NO: 24, SEQ IDNO: 26, and SEQ ID NO: 28. The proton of interest is circled in thepolysaccharide shown above the spectra. The ¹H NMR spectra for a 3-Osulfated product synthesized by enzymes comprising the amino acidsequence of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28 upon reactingwith either PNS and/or NCS are all illustrated. In each of the productspectra, the IdoA_(2S) peak shifts to between approximately 5.0 and 5.05ppm. A similar transition is shown when incubating the natural humansulfotransferase enzyme with the same polysaccharide substrate and PAPS(data not shown).

As shown in FIG. 32 , the region between 4.5 and 3.5 shows several peaksthat similarly shift in response to the addition of the sulfate group tothe 3-O position of a glucosamine residue, all of which correlate to thesame shifts observed upon incubating the natural human 3OST enzyme withthe same commercial heparin substrate and PAPS. Peaks that shift areindicated in curved arrows, and positions of the peaks from 3-O sulfatedpolysaccharides produced by enzymes having the amino acid sequence ofSEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28, are shown with straightarrows. The largest shift occurs for H3 of Glc_(NS3S6S), from 3.7 ppm to4.2 ppm. This results from being closest to the newly added 3-O sulfategroup. Additionally, the H3 proton of Ido_(2S) and H5 of Glc_(NS3S6S)both converge toward a peak at 4.07 ppm, which shows two overlappingpeaks. H4 of Glc_(NS3S6S) shifts moderately downfield from the 3.7 ppmregion to the 3.8 ppm region, and according to references, many peakssuch as H3 & H4 from Glc_(NS6S) and H3, H4, and H5 from GlcA shift fromthe 3.7 ppm region to the 3.6 ppm region.

Example 7: Chemical Synthesis of N-Sulfated Heparosan

A study was conducted in accordance with embodiments of the presentdisclosure to chemically synthesize N-sulfated heparosan that can beutilized as sulfo acceptor polysaccharides with any of the engineeredaryl sulfate-dependent enzymes, particularly either of the engineered2OST enzymes. N-deacetylated heparosan was prepared according to theprotocol described in Balagurunathan, K. et al., above. Particularly,the heparosan that eluted from the DEAE resin was precipitated overnightin ethanol saturated with sodium acetate, at −30° C., before beingresuspended in water and dialyzed within a cellulose dialysis membranehaving a 1,000 Da molecular weight cut-off (MWCO).

To N-deacetylate the heparosan, enough sodium hydroxide pellets (˜4.0 g)were dissolved to make a 2.5 M solution in a 40 mL aliquot of thedialyzed heparosan in water. The solution was incubated at 55° C. for 16hours, with shaking at 100 rpm. The sodium hydroxide within the samplewas then neutralized with acetic acid until the solution reached a pH of˜7.0, and then dialyzed in water overnight within a 1,000 MWCO dialysismembrane.

Subsequent N-sulfation of the N-deacetylated heparosan was accomplishedby adding 100 mg of sodium carbonate and 100 mg of sulfurtrioxide-triethylamine complex, and allowing the composition to incubateat 48° C. until all of the solid was dissolved. The pH of the solutionwas then readjusted to ˜9.5, using acetic acid. After incubation at 48°C. overnight with shaking at 100 rpm, an additional 100 mg of sodiumcarbonate and 100 mg of sulfur trioxide-triethylamine complex was added,before subsequent readjustment of the pH to ˜9.5 using acetic acid. Thesolution was incubated at 48° C. for an additional 24 hours. Thesulfated polysaccharide solution was neutralized with acetic acid to apH of ˜7.0, and dialyzed in water overnight within a 1,000 MWCO dialysismembrane. The dialyzed N-sulfated heparosan was then lyophilized priorto further use. The N-sulfated heparosan was then further purified byloading it onto a Zenix SEC-100 column and eluting it isocratically with0.1 M ammonium acetate, pH 9.0.

The functionalization of the purified heparosan-based polysaccharide wascharacterized by digesting it with a mixture of three carbon-oxygenlyases comprising the amino acid sequences of SEQ ID NO: 30, SEQ ID NO:31, and SEQ ID NO: 32, and analyzing the digested samples using SAX,using a similar procedure described above. As a positive control, thecommercial HD005 disaccharide of Example 2, containing N-sulfatedglucosamine residues, was also analyzed. Representative chromatograms ofboth samples are shown in FIG. 33 . In both chromatograms, a strong peakis present at about 16.5 minutes, indicating that the synthesized samplecontains N-sulfated glucosamine residues.

Example 8: Preparation of an N,2-HS Polysaccharide Product

A study was conducted in accordance with embodiments of the presentdisclosure to synthesize an N,2-HS polysaccharide product using anengineered 2OST, using the N-sulfated heparosan synthesized in Example 7as the sulfo acceptor. In a conical-bottom centrifuge tube, 80 mMaliquots of NCS were dissolved in 50 mM MES pH 7.0, 2 mM CaCl₂). To eachsolution, 2 mg of the enzyme having the sequence of SEQ ID NO: 14, basedon the absorbance of the enzyme sample at 280 nm, was added (about 4mL). 5 mg of the lyophilized N-sulfated heparosan synthesized in Example7 was resuspended in 1 mL of water and added to the reaction mixturecontaining the enzyme and NCS. The entire reaction mixture was thenincubated at 34° C. with shaking at 30 rpm, for 48 hours. A second setof reactions were prepared using the same procedure, except that 2 mg ofa C₅-hexuronyl epimerase comprising the amino acid sequence of SEQ IDNO: 29 was also added to the reaction mixture, prior to incubation.

The sulfated polysaccharide products from both sets of reactions werepurified by first precipitating out the proteins from the reactionmixtures by placing the reaction vessels in boiling water for 10 minutesand centrifuging at high speed to form a pellet. The supernatantcontaining the polysaccharide products was decanted from the pellet anddialyzed in water overnight within a 1,000 MWCO dialysis membrane. Thedialyzed products were then lyophilized for future use.

To characterize the polysaccharide products, lyophilized samples wereresuspended in 400 μL of water, and purified using a Q-Sepharose FastFlow Column (GE Biosciences). Samples were eluted from the column usinga gradient ranging from 0 to 2M NaCl, in 20 mM sodium acetate buffer, pH5.0. Purified polysaccharides were then digested and analyzed by SAXaccording to the procedures in Example 2 above, along with a commercialpolysaccharide, HD002 (Iduron), which contains disaccharides of 2-Osulfated uronic acid and N-sulfated glucosamine. Representativechromatograms of reactions either without or including the epimeraseenzyme are shown in FIG. 34 and FIG. 35 , respectively. In FIG. 34 , thechromatogram for the HD002 disaccharide has a single, sharp peak atabout 21.1 minutes, which correlates to a sharp peak at a nearlyidentical time in the reaction product, indicating the time that anN,2-HS was formed as a result of the reaction. In FIG. 35 , the HD002disaccharide was provided within a mixture containing other disaccharidestandards, with the disaccharide corresponding to HD002 eluting at 20.5minutes, corresponding with the elution time of the HD002 standard inFIG. 34 . The epimerized reaction product has a sharp peak at a nearlyidentical elution time to the HD002 standard, indicating that an N,2-HSproduct was formed as a result of the reaction.

Example 9: Preparation of an N,2,6-HS Product

A study was conducted in accordance with embodiments of the presentdisclosure to synthesize an N,2,6-HS product using the procedure ofExample 8, except that the N,2-HS product of Example 8 was used as thesulfo acceptor polysaccharide, and the engineered 6OST having the aminoacid sequence of SEQ ID NO: 18 was used as the enzyme.

Representative chromatograms of the sulfated polysaccharide product anda mixture of commercial disaccharides are shown in FIG. 36 . Thechromatogram of the commercial mixture exhibits a peak at about 23.7minutes and correlates to HD001 (Iduron), which consists ofdisaccharides of 2-O sulfated uronic acid and N-, 6-O sulfatedglucosamine, while the reaction product exhibits a similar peak at 23.4minutes, indicating that an N,2,6-HS was formed as a result of thereaction. Other peaks present within the N,2,6-HS product includeundigested polysaccharide (2.5 min), unsubstituted uronic acid andN-acetylated glucosamine (5.5 min), and unsubstituted uronic acid andN-, 6-O sulfated glucosamine.

Example 10: Preparation of an N,2,3,6-HS Product

A study was conducted in accordance with embodiments of the presentdisclosure to synthesize a sulfated polysaccharide product comprisingN-, 6-O, 3-O sulfated glucosamine and 2-O sulfated hexuronic acidresidues, using the procedure of Example 8, except that the chemicallysynthesized N-, 2-O, 6-O sulfated polysaccharide of Example 9 is used asthe sulfo acceptor polysaccharide, and an engineered 3-Osulfotransferase enzyme having the amino acid sequence of SEQ ID NO: 28is used as the sulfotransferase enzyme.

Sulfated polysaccharide products were digested and analyzed using LCMSto confirm the production of an N,2,3,6-HS product. To facilitate studyusing LCMS, sulfated polysaccharide products of the SEQ ID NO: 28sulfotransferase enzyme were isolated and derivatized with aniline tags,according to the procedures described in Lawrence, R., et al., (2008) J.Biol. Chem. 283 (48):33674-33684, the disclosure of which isincorporated by reference in its entirety. Briefly, some GAGs, includingcommercial heparin and other N,2,3,6-HS polysaccharides, can bequantified and compared ratiometrically using LCMS by chemicallymodifying the sulfated product. Lawrence, R., et al., describes thetagging of the reducing end of lyase-generated disaccharides andtetrasaccharides with [¹²C₆]- and [¹³C₆]-aniline and propionylation ofN-unsubstituted glucosamine residues. Isotopic tagging of thedisaccharides and tetrasaccharides has no effect on the chromatographicretention times, but can be discriminated using mass spectroscopy.

Sulfated disaccharide and tetrasaccharide products were prepared byanion exchange chromatography, as described in Example 8, and digestionwith a mixture of three carbon-oxygen lyases comprising the amino acidsequences of SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, asdescribed above in Example 7. 1 pmol to 10 nmol of the digested sampleswere transferred to 1.5-ml microcentrifuge tubes and dried down in acentrifugal evaporator. [¹²C₆]-aniline or [¹³C₆]-aniline (15 μl, 165μmol) and 15 μl of 1 M NaCNBH₃ freshly prepared in dimethylsulfoxide:acetic acid (7:3, v/v) were added to each sample. Reactionswere carried out at 65° C. for 4 h, or alternatively at 37° C. for 16 h,and then dried in a centrifugal evaporator.

Unsubstituted amines were reacted with propionic anhydride. Driedsamples were reconstituted in 20 μl of 50% methanol, and 3 μl ofpropionic anhydride (23.3 μmol) was added. Reactions were carried out atroom temperature for 2 h. Acylated samples were subsequentlyaniline-tagged as described above.

A quadrupole ion trap Liquid Chromatograph Mass Spectrometer with anelectrospray ionization source, similar to the Shimadzu LCMS-8050 massspectrometer described in Example 3, was used for disaccharide analysis.Derivatized and non-derivatized disaccharide residues were separated ona C18 reversed-phase column with the ion pairing agent dibutylamine(DBA). The isocratic steps were: 100% buffer A (8 mm acetic acid, 5 mmDBA) for 10 min, 17% buffer B (70% methanol, 8 mm acetic acid, 5 mm DBA)for 15 min; 32% buffer B for 15 min, 40% buffer B for 15 min, 60% bufferB for 15 min; 100% buffer B for 10 min; and 100% buffer A for 10 min.Generally, mass spectra for samples containing 3-O sulfated product areexpected to generate m/z peaks corresponding to tetrasaccharides thatare resistant to digestion by the carbon-oxygen lyases, as describedabove in Example 5. Tetrasaccharides that can be produced include, butare not limited to: 4,5-unsaturated uronic acid-N-acetylated, 6-Osulfated glucosamine-glucuronic acid-N-sulfated, 3-O sulfatedglucosamine (ΔU-A_(NAc6S)-G-A_(NS3S)); 4,5-unsaturated uronicacid-N-acetylated, 6-O sulfated glucosamine-glucuronic acid-N-sulfated,3-O sulfated, 6-O sulfated glucosamine (AU-A_(NAc6S)-G-A_(NS3S6S));4,5-unsaturated uronic acid-N-sulfated, 6-O sulfatedglucosamine-glucuronic acid-N-sulfated, 3-O sulfated, 6-O sulfatedglucosamine (ΔU-A_(NS6S)-G-A_(NS3S6S)); 4,5-unsaturated, 2-O sulfateduronic acid-N-sulfoglucosamine-glucuronic acid-N-sulfated, 3-O sulfated,6-O sulfated glucosamine (ΔU2S-A_(NS)-G-A_(NS3S6S)); and4,5-unsaturated, 2-O sulfated uronic acid-N-sulfated, 6-O sulfatedglucosamine-glucuronic acid-N-sulfated, 3-O sulfated, 6-O sulfatedglucosamine (ΔU2S-A_(NS6S)-G-A_(NS3S6S)). In particular, LCMS of thedigested polysaccharide samples collected from the reaction with the SEQID NO: 28 sulfotransferase enzyme generated mass spectra (not shown)with m/z peaks corresponding to the ΔU-A_(NAc6S)-G-A_(NS3S6S)(m/z=1036), ΔU-A_(NS6S)-G-A_(NS3S6S) (m/z=1074), andΔU2S-A_(NS6S)-G-A_(NS3S6S) (m/z=1154) tetrasaccharides, indicating thatthe N,2,3,6-HS product was produced by the reaction with the SEQ ID NO:28 engineered sulfotransferase enzyme.

Example 11: Confirmation of Anticoagulant Activity of the N,2,3,6-HSProduct

A study is conducted in accordance with embodiments of the presentdisclosure to determine whether 3-O sulfated polysaccharide productsproduced in Example 10 have a binding affinity to antithrombin using aprocedure similar to Meneghetti, G., et al. (2017) Org. Biomol. Chem.15:6792-6799). It is expected that melting curves of antithrombin in thepresence of the 3-O sulfated polysaccharide products produced in Example10 will demonstrate a higher melting temperature than antithrombinalone, indicating that the 3-O sulfated polysaccharide product producedin Example 10 comprises the structure of Formula I.

Example 12: Determination of Engineered Aryl Sulfate-Dependent Mutantsof Other EC 2.8.2.8 Enzymes

A study is conducted in accordance with embodiments of the presentdisclosure to engineer additional aryl sulfate-dependent NST enzymes. Asdescribed above, the aryl sulfate-dependent NST enzymes having the aminoacid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, and SEQ ID NO: 12 have been engineered to be mutantsof the N-sulfotransferase domain of the human glucosaminylN-deacetylase/N-sulfotransferase enzyme (see entrysp|P52848|NDST_1_HUMAN, in FIG. 3 above), which is a member of enzymeclass EC 2.8.2.8. By generating and analyzing a multiple sequencealignment that includes both the amino acid sequences of theN-sulfotransferase domain of one or more of the other glucosaminylN-deacetylase/N-sulfotransferase enzymes within EC 2.8.2.8 as well asthe amino acid sequences of aryl sulfate-dependent NST enzymes havingthe amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, and/or SEQ ID NO: 12, mutations in theamino acid sequences in the engineered NST enzymes can be observedrelative to the amino acid sequences of the natural EC 2.8.2.8 enzymeswithin the same alignment. Upon selecting the amino acid sequence of theN-sulfotransferase domain of a natural 2.8.2.8 enzyme that is not thehuman glucosaminyl N-deacetylase/N-sulfotransferase enzyme, mutationsthat are present within the amino acid sequences of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and/or SEQ ID NO: 12can be engineered into the natural sequence in order to form additionalmutants that can have aryl sulfate-dependent sulfotransferase activity.

As a non-limiting example, the amino acid sequence encoding for theN-sulfotransferase domain of the pig glucosaminylN-deacetylase/N-sulfotransferase enzyme (entry tr|M3V841|M3V841_PIG, asillustrated in the sequence alignment in FIG. 3 ), is aligned with theamino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, and SEQ ID NO: 12. Amino acid mutations that arepresent in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, and SEQ ID NO: 12 are engineered into their equivalentpositions within the amino acid sequence of the N-sulfotransferasedomain of the pig N-deacetylase/N-sulfotransferase enzyme, in order togenerate the mutant amino acid sequences SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40,respectively. Enzymes comprising the amino acid sequences of SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQID NO: 40, respectively, will be utilized in Example 13 and Example 14,below. However, a person skilled in the art would appreciate that thesame procedure can be applied to generate mutants of theN-sulfotransferase domain, or the entire enzyme, with respect to any ofthe other glucosaminyl natural N-deacetylase/N-sulfotransferase enzymeswithin the EC 2.8.2.8 enzyme class, and that those are omitted forclarity.

Example 13: Expression and Purification of Engineered ArylSulfate-Dependent EC 2.8.2.8 Mutants

A study is conducted in accordance with embodiments of the presentdisclosure to determine whether genes encoding for engineered NSTenzymes having the amino acid sequences SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40,respectively, can be transformed into host cells, and that enzymescomprising each of those amino acid sequences can be subsequentlyexpressed, isolated, and purified according to the procedure of Example1, above. Codon-optimized nucleotide sequences are determined thatencode for enzymes having the amino acid sequences of SEQ ID NO: 35, SEQID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO:40, respectively, based on the desired expression host. Uponsynthesizing or inserting those genes within a suitable expressionvector, it is expected that genes encoding for each of the amino acidsequences SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,SEQ ID NO: 39, and SEQ ID NO: 40, respectively, will be transformed intohost cells, and that enzymes containing those sequences will besubsequently expressed, isolated, and purified in a sufficient quantityand purity to determine aryl sulfate-dependent NST activity.

Example 14: Sulfotransferase Activity of EC 2.8.2.8 Mutants

A study is conducted in accordance with embodiments of the presentdisclosure to determine whether mutant enzymes comprising the sequencesof SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, and SEQ ID NO: 40, respectively, are active sulfotransferases,using the procedures of Example 2. It is expected that SAX studies willconfirm the presence of N-sulfated polysaccharide products formed as aresult of reacting N-deacetylated heparosan and an aryl sulfate compoundwith each of the engineered enzymes comprising the sequences of SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, orSEQ ID NO: 40, respectively.

Example 15: Determination of Engineered Aryl Sulfate-Dependent Mutantsof Other 2OST Enzymes within EC 2.8.2.-

A study is conducted in accordance with embodiments of the presentdisclosure to engineer additional aryl sulfate-dependent 2OST enzymes.As described above, the aryl sulfate-dependent 2OST enzymes having theamino acid sequences of SEQ ID NO: 14 and SEQ ID NO: 16 have beenengineered to be mutants of the chicken 2OST enzyme (see entrysp|Q76KB1|HS2ST_CHICK, in FIGS. 14A-14D, above), which is a member ofenzyme class EC 2.8.2.-. By generating and analyzing a multiple sequencealignment that includes both the amino acid sequences of one or more ofthe other 2OST enzymes within EC 2.8.2.-, as well as the amino acidsequences of aryl sulfate-dependent 2OST enzymes having the amino acidsequences of SEQ ID NO: 14 and/or SEQ ID NO: 16, mutations in the aminoacid sequences in the engineered 2OST enzymes can be observed relativeto the amino acid sequences of the natural 2OST enzymes within the samealignment. Upon selecting the amino acid sequence of a natural 2OSTenzyme that is not the chicken 2OST enzyme, mutations that are presentwithin the amino acid sequences of SEQ ID NO: 14 and/or SEQ ID NO: 16can be engineered into the natural sequence in order to form additionalmutants that can have aryl sulfate-dependent sulfotransferase activity.

As a non-limiting example, the amino acid sequence encoding for thehuman 2OST enzyme (entry sp|Q7LGA3|HS2ST_HUMAN, as illustrated in thesequence alignment in FIGS. 14A-14D), is aligned with the amino acidsequences of SEQ ID NO: 14 and SEQ ID NO: 16. Amino acid mutations thatare present in SEQ ID NO 14 and SEQ ID NO: 16 are engineered into theirequivalent positions within the amino acid sequence of the human 2OSTenzyme, in order to generate the mutant amino acid sequences SEQ ID NO:41 and SEQ ID NO: 42, respectively. Enzymes comprising the amino acidsequences of SEQ ID NO: 41 and SEQ ID NO: 42, respectively, will beutilized in Example 16 and Example 17, below. However, a person skilledin the art would appreciate that the same procedure can be applied togenerate aryl sulfate-dependent mutants with respect to any of the other2OST enzymes within the EC 2.8.2.- enzyme class, and that those areomitted for clarity.

Example 16: Expression and Purification of EC 2.8.2.- Mutants Having2OST Activity

A study is conducted in accordance with embodiments of the presentdisclosure to determine whether genes encoding for engineered 2OSTenzymes having the amino acid sequences SEQ ID NO: 41 and SEQ ID NO: 42,respectively, can be transformed into host cells, and that enzymescomprising each of those amino acid sequences can be subsequentlyexpressed, isolated, and purified according to the procedure of Example1, above. Codon-optimized nucleotide sequences are determined thatencode for enzymes having the amino acid sequences of SEQ ID NO: 41 andSEQ ID NO: 42, respectively, based on the desired expression host. Uponsynthesizing or inserting those genes within a suitable expressionvector, it is expected that genes encoding for each of the amino acidsequences SEQ ID NO: 41 and SEQ ID NO: 42, respectively, will betransformed into host cells, and that enzymes containing those sequenceswill be subsequently expressed, isolated, and purified in a sufficientquantity and purity to determine aryl sulfate-dependent 2OST activity.

Example 17: 2OST Activity of EC 2.8.2.- Mutants

A study is conducted in accordance with embodiments of the presentdisclosure to determine whether mutant enzymes comprising the sequencesof SEQ ID NO: 41 and SEQ ID NO: 42, respectively, are activesulfotransferases, using the procedures of Example 3. It is expectedthat MS studies will confirm the presence of N,2-HS products formed as aresult of reacting an N-sulfated heparosan-based polysaccharide and anaryl sulfate compound with each of the engineered enzymes comprising thesequences of SEQ ID NO: 41 and SEQ ID NO: 42, respectively. It is alsoexpected that both enzymes will be active with heparosan-basedpolysaccharides comprising either or both of Formula IV or Formula V.

Example 18: Determination of Engineered Aryl Sulfate-Dependent Mutantsof Other 6OST Enzymes within EC 2.8.2.-

A study is conducted in accordance with embodiments of the presentdisclosure to engineer additional aryl sulfate-dependent 6OST enzymes.As described above, the aryl sulfate-dependent 6OST enzymes having theamino acid sequences of SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22have been engineered to be mutants of isoform 1 of the mouse 6OST enzyme(see entry Q9QYK5|H6ST1_MOUSE, in FIGS. 18A-18C, above), which is amember of enzyme class EC 2.8.2.-. By generating and analyzing amultiple sequence alignment that includes both the amino acid sequencesof one or more of the other 6OST enzymes within EC 2.8.2.-, as well asthe amino acid sequences of aryl sulfate-dependent 6OST enzymes havingthe amino acid sequences of SEQ ID NO: 18, SEQ ID NO: 20, and/or SEQ IDNO: 22, mutations in the amino acid sequences in the engineered 6OSTenzymes can be observed relative to the amino acid sequences of thenatural 6OST enzymes within the same alignment. Upon selecting the aminoacid sequence of a natural 6OST enzyme that is not the mouse 6OSTenzyme, mutations that are present within the amino acid sequences ofSEQ ID NO: 18, SEQ ID NO: 20, and/or SEQ ID NO: 22 can be engineeredinto the natural sequence in order to form additional mutants that canhave aryl sulfate-dependent sulfotransferase activity.

As a non-limiting example, the amino acid sequence encoding for the pig6OST enzyme (entry I3LAM6|I3LAM6_PIG, as illustrated in the sequencealignment in FIGS. 18A-18C), is aligned with the amino acid sequences ofSEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. Amino acid mutationsthat are present in SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22 areengineered into their equivalent positions within the amino acidsequence of the pig 6OST enzyme, in order to generate mutant amino acidsequences. Generated mutant amino acid sequences corresponding toresidues 67-377 of the pig 6OST enzyme, as illustrated in FIGS. 18A-18C,are disclosed as SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47,respectively. Generated mutant amino acid sequences corresponding to thefull-length amino acid sequence for the pig 6OST enzyme (not shown inFIGS. 18A-18C) are disclosed as SEQ ID NO: 48, SEQ ID NO: 49, and SEQ IDNO: 50, respectively.

In another non-limiting example, the full-length amino acid sequenceencoding for the encoding for isoform 3 of the mouse 6OST enzyme (entryQ9QYK4|H6HS3_MOUSE, a truncated sequence for which is illustrated in thesequence alignment in FIGS. 18A-18C) is aligned with the amino acidsequences of SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. Amino acidmutations that are present in SEQ ID NO: 18, SEQ ID NO: 20, and SEQ IDNO: 22 are engineered into their equivalent positions within the aminoacid sequence of isoform 3 of the mouse 6OST enzyme, in order togenerate mutant amino acid sequences. The generated full-length aminoacid sequences are disclosed as SEQ ID NO: 59, SEQ ID NO: 60, and SEQ IDNO: 61, respectively. Enzymes comprising the amino acid sequences of SEQID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61,respectively, will be utilized in Example 19 and Example 20, below.However, a person skilled in the art would appreciate that the sameprocedure can be applied to generate aryl sulfate-dependent mutants withrespect to any of the other 6OST enzymes within the EC 2.8.2.-enzymeclass, and that those are omitted for clarity.

Example 19: Expression and Purification of EC 2.8.2.- Mutants Having6OST Activity

A study is conducted in accordance with embodiments of the presentdisclosure to determine whether genes encoding for engineered 6OSTenzymes having the amino acid sequences SEQ ID NO: 45, SEQ ID NO: 46,SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:59, SEQ ID NO: 60, and SEQ ID NO: 61, respectively, can be transformedinto host cells, and that enzymes comprising each of those amino acidsequences can be subsequently expressed, isolated, and purifiedaccording to the procedure of Example 1, above. Codon-optimizednucleotide sequences are determined that encode for enzymes having theamino acid sequences of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60,and SEQ ID NO: 61, respectively, based on the desired expression host.Upon synthesizing or inserting those genes within a suitable expressionvector, it is expected that genes encoding for each of the amino acidsequences SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48,SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ IDNO: 61, respectively, will be transformed into host cells, and thatenzymes containing those sequences will be subsequently expressed,isolated, and purified in a sufficient quantity and purity to determinearyl sulfate-dependent 6OST activity.

Example 20: 6OST Activity of EC 2.8.2.- Mutants

A study is conducted in accordance with embodiments of the presentdisclosure to determine whether mutant enzymes comprising the sequencesof SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61,respectively, are active sulfotransferases, using the procedures ofExample 4. It is expected that MS studies will confirm the presence ofN,2,6-HS products formed as a result of reacting an N,2-HSpolysaccharide and an aryl sulfate compound with each of the engineeredenzymes comprising the sequences of SEQ ID NO: 45, SEQ ID NO: 46, SEQ IDNO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQID NO: 60, and SEQ ID NO: 61, respectively.

Example 21: Determination of Engineered Aryl Sulfate-Dependent Mutantsof Other 3OST Enzymes within EC 2.8.2.23

A study is conducted in accordance with embodiments of the presentdisclosure to engineer additional aryl sulfate-dependent 3OST enzymes.As described above, the aryl sulfate-dependent 3OST enzymes having theamino acid sequences of SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28have been engineered to be mutants of isoform 1 of the human 3OST enzyme(see entry sp|O14792|HS3S1_HUMAN, in FIGS. 23A-23C, above), which is amember of enzyme class EC 2.8.2.23. By generating and analyzing amultiple sequence alignment that includes both the amino acid sequencesof one or more of the other 3OST enzymes within EC 2.8.2.23, as well asthe amino acid sequences of aryl sulfate-dependent 3OST enzymes havingthe amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 26, and/or SEQ IDNO: 28, mutations in the amino acid sequences in the engineered 3OSTenzymes can be observed relative to the amino acid sequences of thenatural 3OST enzymes within the same alignment. Upon selecting the aminoacid sequence of a natural 3OST enzyme that is not the human 3OSTenzyme, mutations that are present within the amino acid sequences ofSEQ ID NO: 24, SEQ ID NO: 26, and/or SEQ ID NO: 28 can be engineeredinto the natural sequence in order to form additional mutants that canhave aryl sulfate-dependent sulfotransferase activity.

As a non-limiting example, the amino acid sequence encoding for isoform1 of the pig 3OST enzyme (entry tr|I3LHH5|I3LHH5_PIG, as illustrated inthe sequence alignment in FIGS. 23A-23C), is aligned with the amino acidsequences of SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28. Amino acidmutations that are present in SEQ ID NO: 24, SEQ ID NO: 26, and SEQ IDNO: 28 are engineered into their equivalent positions within the aminoacid sequence of the pig 3OST enzyme, in order to the generate mutantamino acid sequences SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54,respectively.

In another non-limiting example, the full-length amino acid sequenceencoding for the encoding for isoform 5 of the mouse 3OST enzyme (notshown in FIGS. 18A-18C) is aligned with the amino acid sequences of SEQID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28. Amino acid mutations thatare present in SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28 areengineered into their equivalent positions within the amino acidsequence of isoform 5 of the mouse 3OST enzyme, in order to generatemutant amino acid sequences. The generated full-length amino acidsequences are disclosed as SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO:58, respectively.

Enzymes comprising the amino acid sequences of SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58respectively, will be utilized in Example 22 and Example 23, below.However, a person skilled in the art would appreciate that the sameprocedure can be applied to generate aryl sulfate-dependent mutants withrespect to any of the other 3OST enzymes within the EC 2.8.2.23 enzymeclass, and that those are omitted for clarity.

Example 22: Expression and Purification of EC 2.8.2.23 Mutants Having3OST Activity

A study is conducted in accordance with embodiments of the presentdisclosure to determine whether genes encoding for engineered 3OSTenzymes having the amino acid sequences SEQ ID NO: 52, SEQ ID NO: 53,SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58,respectively, can be transformed into host cells, and that enzymescomprising each of those amino acid sequences can be subsequentlyexpressed, isolated, and purified according to the procedure of Example1, above. Codon-optimized nucleotide sequences are determined thatencode for enzymes having the amino acid sequences of SEQ ID NO: 52, SEQID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO:58, respectively, based on the desired expression host. Uponsynthesizing or inserting those genes within a suitable expressionvector, it is expected that genes encoding for each of the amino acidsequences SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 57, and SEQ ID NO: 58, respectively, will be transformed intohost cells, and that enzymes containing those sequences will besubsequently expressed, isolated, and purified in a sufficient quantityand purity to determine aryl sulfate-dependent 3OST activity.

Example 23: 3OST Activity of EC 2.8.2.23 Mutants

A study is conducted in accordance with embodiments of the presentdisclosure to determine whether mutant enzymes comprising the sequencesof SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ IDNO: 57, and SEQ ID NO: 58, respectively, are active sulfotransferases,using the procedures of Example 5 and/or Example 6. It is expected thatMS and/or NMR studies will confirm the presence of N,2,3,6-HS productsformed as a result of reacting an N,2,6-HS polysaccharide and an arylsulfate compound with each of the engineered enzymes comprising thesequences of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 57, and SEQ ID NO: 58, respectively.

Because the instant application is a continuation application, to theextent any amendments, characterizations, or other assertions previouslymade (in any related patent applications or patents, including anyparent, sibling, or child) with respect to any art, prior or otherwise,could be construed as a disclaimer of any subject matter supported bythe present disclosure of this application, Applicant hereby rescindsand retracts such disclaimer. Applicant also respectfully submits thatany prior art previously considered in any related patent applicationsor patents, including any parent, sibling, or child, may need to bere-visited.

We claim:
 1. A method of enzymatically synthesizing an N-, 2-O, 3-O, 6-Osulfated, heparan sulfate (N,2,3,6-HS) product, the method comprisingthe following steps: (a) providing a starting polysaccharide mixturecomprising N-sulfated heparosan; (b) combining the startingpolysaccharide reaction mixture with a reaction mixture comprising afirst sulfo group donor and a first sulfotransferase enzyme selectedfrom the group consisting of a hexuronyl 2-O sulfotransferase enzyme(2OST) enzyme and a glucosaminyl 6-O sulfotransferase enzyme (6OST)enzyme, to form a first sulfated polysaccharide product; (c) combiningthe first sulfated polysaccharide product with a reaction mixturecomprising a second sulfo group donor and a second sulfotransferaseenzyme, wherein the second sulfotransferase enzyme is the enzyme thatwas not selected in step (b), to form a second sulfated polysaccharideproduct; (d) combining the second sulfated polysaccharide product with areaction mixture comprising a third sulfo group donor and a glucosaminyl3-O sulfotransferase enzyme (3OST) enzyme, to form the N,2,3,6-HSproduct; wherein the sulfotransferase enzyme in each of steps (b), (c),and (d) is selected from the group consisting of a naturalsulfotransferase enzyme and an engineered sulfotransferase enzyme,wherein a natural sulfotransferase enzyme has sulfotransferase activitywith 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as a sulfo group donorand heparan sulfate as a sulfo group acceptor, and when a naturalsulfotransferase enzyme is selected in any of steps (b), (c), or (d),the sulfo group donor is PAPS; wherein an engineered sulfotransferaseenzyme has sulfotransferase activity with an aryl sulfate compound as asulfo group donor and heparan sulfate as a sulfo group acceptor, andwhen an engineered sulfotransferase enzyme is selected in any of steps(b), (c), or (d), the sulfo group donor is an aryl sulfate compound; andwherein at least one of the sulfotransferase enzymes selected in steps(b), (c), and (d) is an engineered sulfotransferase enzyme.
 2. Themethod of claim 1, wherein the first sulfotransferase enzyme is the 2OSTenzyme, and the second sulfotransferase enzyme is the 6OST enzyme. 3.The method of claim 2, wherein the 3OST enzyme is an engineeredsulfotransferase enzyme.
 4. The method of claim 3, wherein the thirdsulfo group donor is selected from the group consisting of p-nitrophenylsulfate (PNS) and 4-nitrocatechol sulfate (NCS).
 5. The method of claim4, wherein the 3OST enzyme comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ IDNO: 56, SEQ ID NO: 57, and SEQ ID NO:
 58. 6. The method of claim 3,wherein at least one of the 2OST enzyme and the 6OST enzyme is anengineered sulfotransferase enzyme.
 7. The method of claim 6, whereinthe 6OST enzyme is an engineered sulfotransferase enzyme.
 8. The methodof claim 7, wherein the second sulfo group donor is selected from thegroup consisting of PNS and NCS.
 9. The method of claim 8, wherein the6OST enzyme comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, andSEQ ID NO:
 61. 10. The method of claim 6, wherein the 2OST enzyme is anengineered sulfotransferase enzyme.
 11. The method of claim 10, whereinthe first sulfo group donor is selected from the group consisting of PNSand NCS.
 12. The method of claim 11, wherein the 2OST enzyme comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 41, and SEQ ID NO:
 42. 13. The method ofclaim 2, wherein the first sulfotransferase reaction mixture furthercomprises a glucuronyl C₅-epimerase enzyme.
 14. The method of claim 1,wherein at least a portion of the glucosamine residues within theN-sulfated heparosan are N-acetylated.
 15. The method according to claim1, wherein the N,2O,3O,6O-HS product is a heparin product, the heparinproduct comprising polysaccharides having a sequence motif having thestructure of Formula I, below:

wherein X is either a sulfo group or an acetate group and Y is either asulfo group or a hydroxyl group.
 16. The method according to claim 15,wherein the heparin product has anticoagulant activity and is apolydisperse mixture of polysaccharides having a weight averagemolecular weight, M _(w), of at least 1,000 Da.
 17. The method accordingto claim 15, wherein the heparin product is further fractionated to forma low-molecular weight heparin (LMWH) product, the method furthercomprising the steps: (e) providing one or more depolymerization agents;and (f) treating the heparin product with the one or moredepolymerization agents for a time sufficient to depolymerize at leastsome of the polysaccharides within the heparin product, thereby formingthe LMWH product.
 18. The method according to claim 17, wherein one ormore depolymerization agents are selected from the group consisting of aβ-elimination agent, a deamination agent, and an oxidation agent,wherein the β-elimination agent is selected from the group consistingof: a carbon-oxygen lyase reaction mixture comprising at least onecarbon-oxygen lyase enzyme, and one or more basic compounds selectedfrom the group consisting of sodium hydroxide, a quaternary ammoniumhydroxide, and a phosphazene base, wherein the deamination agent isselected from the group consisting of isoamyl nitrate and nitrous acid;and wherein the oxidizing agent is selected from the group consisting ofa peroxide or superoxide compound.
 19. The method according to claim 18,wherein the M _(w) of the LMWH product is in a range from at least about2,000 Da, and up to about 12,000 Da.
 20. The method according to claim19, wherein the LMWH product has anticoagulant activity.